Methods of treating colorectal cancer

ABSTRACT

This disclosure provides methods for treating a tumor derived from a colorectal cancer exhibiting a high degree of microsatellite instability in a subject comprising administering to the subject an anti-PD-1 antibody. In some embodiments, the method further comprises administering an anti-CTLA-4 antibody. In some embodiments, the colorectal cancer is rectal cancer, colon cancer, or any combination thereof.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.16/306,290, which is the U.S. national phase entry of InternationalPatent Application No. PCT/US2017/035822, filed Jun. 2, 2017, whichclaims the benefit of U.S. Provisional Application No. 62/345,662, filedJun. 3, 2016, each of which is incorporated herein by reference in itsentirety.

FIELD OF THE DISCLOSURE

This disclosure relates to methods for treating a tumor derived from acolorectal cancer exhibiting a high degree of microsatellite instability(MSI-H) in a subject in need thereof comprising administering to thesubject an anti-Programmed Death-1 (PD-1) antibody. In some embodiments,the anti-PD-1 antibody is administered in combination with ananti-Cytotoxic T-Lymphocyte Antigen-4 (CTLA-4) antibody. In someembodiments, the colorectal cancer is rectal cancer, colon cancer, orany combination thereof.

BACKGROUND OF THE DISCLOSURE

Human cancers harbor numerous genetic and epigenetic alterations,generating neoantigens potentially recognizable by the immune system(Sjoblom et al. (2006) Science 314:268-74). The adaptive immune system,comprised of T and B lymphocytes, has powerful anti-cancer potential,with a broad capacity and exquisite specificity to respond to diversetumor antigens. Further, the immune system demonstrates considerableplasticity and a memory component. The successful harnessing of allthese attributes of the adaptive immune system would make immunotherapyunique among all cancer treatment modalities.

Until recently, cancer immunotherapy had focused substantial effort onapproaches that enhance anti-tumor immune responses by adoptive-transferof activated effector cells, immunization against relevant antigens, orproviding non-specific immune-stimulatory agents such as cytokines. Inthe past decade, however, intensive efforts to develop specific immunecheckpoint pathway inhibitors have begun to provide newimmunotherapeutic approaches for treating cancer, including thedevelopment of an antibody (Ab), ipilimumab (YERVOY®), that binds to andinhibits CTLA-4 for the treatment of patients with advanced melanoma(Hodi et al. (2010)N Engl J Med 363:711-23) and the development ofantibodies such as nivolumab and pembrolizumab (formerly lambrolizumab;USAN Council Statement, 2013) that bind specifically to the ProgrammedDeath-1 (PD-1) receptor and block the inhibitory PD-1/PD-1 ligandpathway (Topalian et al. N Engl J Med 366:2443-54 (2012a); Topalian etal. Curr Opin Immunol 24:207-12 (2012b); Topalian et al. J Clin Oncol32(10):1020-30 (2014); Hamid et al. N Engl J Med 369:134-144 (2013);Hamid and Carvajal Expert Opin Biol Ther 13(6):847-61 (2013); McDermottand Atkins Cancer Med 2(5):662-73(2013)).

Nivolumab (formerly designated 5C4, BMS-936558, MDX-1106, or ONO-4538)is a fully human IgG4 (S228P) PD-1 immune checkpoint inhibitor antibodythat selectively prevents interaction with PD-1 ligands (PD-L1 andPD-L2), thereby blocking the down-regulation of antitumor T-cellfunctions (U.S. Pat. No. 8,008,449; Wang et al., (2014) Cancer ImmunolRes 2:846-56). Nivolumab has shown activity in a variety of advancedsolid tumors including renal cell carcinoma (renal adenocarcinoma, orhypernephroma), melanoma, and non-small cell lung cancer (NSCLC)(Topalian et al., (2012) N Engl J Med 366:2443-54; Topalian et al.,(2014) J Clin Oncol 32:1020-30; Drake et al., (2013) BJU Int 112:1-17;Ansell et al., (2015) Blood 126:583 [Abstract]; PCT Publication No. WO2013/173223).

Colorectal cancer is the third most common type of cancer in both menand women in the U.S. (See http://www.cancer.gov/types/colorectal, lastvisited Dec. 9, 2015). Most colorectal cancers are adenocarcinomas. Somecolorectal cancers are associated with a high degree of microsatelliteinstability (MSI-H), which results from impaired DNA mismatch repair. Inparticular, patients with DNA mismatch repair-deficient/microsatelliteinstability-high (dMMR/MSI-H) metastatic colorectal cancer (mCRC) (˜5%of patients) are less likely to benefit from conventional chemotherapythan patients with proficient MMR (pMMR) mCRC. dMMR-MSI-H CRC isassociated with a high mutational burden and is particularly susceptibleto immune checkpoint inhibitor blockade.

Targeted therapy of multiple non-redundant molecular pathways regulatingimmune responses may enhance antitumor immunotherapy. However, not alltherapies are acceptable. There remains a need for therapies with anacceptable safety profile and high efficacy that enhance antitumorimmune responses compared to monotherapy and other immunotherapycombinations.

SUMMARY OF THE DISCLOSURE

The present disclosure relates to a method for treating a subjectafflicted with a tumor derived from a colorectal cancer exhibiting ahigh degree of microsatellite instability (MSI-H) comprisingadministering to the subject a therapeutically effective amount of anantibody or an antigen-binding portion thereof that binds specificallyto Programmed Death-1 receptor (PD-1) and inhibits PD-1 activity(“anti-PD-1 antibody”). In some embodiments, the method furthercomprises administering a therapeutically effective amount of anantibody or antigen-binding portion thereof that specifically binds toCytotoxic T-Lymphocyte Antigen-4 (CTLA-4) and inhibits CTLA-4 activity(“anti-CTLA-4 antibody”).

In some embodiments, the colorectal cancer is rectal cancer, coloncancer, or any combination thereof. In one embodiment, the administeringtreats the colorectal cancer.

In some embodiments, the anti-PD-1 antibody cross-competes withnivolumab for binding to human PD-1. In some embodiments, the anti-PD-1antibody binds to the same epitope as nivolumab. In certain embodiments,the anti-PD-1 antibody is a chimeric, humanized or human monoclonalantibody or a portion thereof. In some embodiments, the anti-PD-1antibody comprises a heavy chain constant region which is of a humanIgG1 or IgG4 isotype. In one embodiment, the anti-PD-1 antibody isnivolumab. In another embodiment, the anti-PD-1 antibody ispembrolizumab.

In certain embodiments, the anti-PD-1 antibody is administered at a doseranging from at least about 0.1 mg/kg to at least about 10.0 mg/kg bodyweight once about every 1, 2 or 3 weeks. In one embodiment, theanti-PD-1 antibody (e.g., nivolumab) is administered at a dose of atleast about 1 mg/kg body weight once about every 3 weeks. In anotherembodiment, the anti-PD-1 antibody (e.g., nivolumab) is administered ata dose of at least about 3 mg/kg body weight once about every 3 weeks.In yet another embodiment, the anti-PD-1 antibody (e.g., nivolumab) isadministered at a dose of at least about 3 mg/kg body weight once aboutevery 2 weeks. In other embodiments, the anti-PD-1 antibody (e.g.,pembrolizumab) is administered at a dose of at least about 200 mg every3 weeks or 2 mg/kg (up to 200 mg) every three weeks. In someembodiments, the anti-PD-1 antibody (e.g., avelumab) is administered ata dose of 10 mg/kg every two weeks. In certain embodiments, theanti-PD-1 antibody is administered at a flat dose. In embodiments, theanti-PD-1 antibody is administered at a flat dose of at least about 200mg, at least about 220 mg, at least about 240 mg, at least about 260 mg,at least about 280 mg, at least about 300 mg, at least about 320 mg, atleast about 340 mg, at least about 360 mg, at least about 380 mg, atleast about 400 mg, at least about 420 mg, at least about 440 mg, atleast about 460 mg, at least about 480 mg, at least about 500 mg, atleast about 550 mg, at least about 600 mg, at least about 650 mg, atleast 700 mg, at least 750 mg, or at least 800 mg. In some embodiments,the anti-PD-1 antibody is administered for as long as clinical benefitis observed or until unmanageable toxicity or disease progressionoccurs.

In some embodiments, the anti-CTLA-4 antibody cross-competes withipilimumab for binding to human CTLA-4. In one embodiment, theanti-CTLA-4 antibody binds to the same epitope as ipilimumab. In someembodiments, the anti-CTLA-4 antibody is a chimeric, humanized or humanmonoclonal antibody or a portion thereof. In certain embodiments, theanti-CTLA-4 antibody comprises a heavy chain constant region which is ofa human IgG1 or IgG2 isotype. In one embodiment, the anti-CTLA-4antibody is ipilimumab. In another embodiment, the anti-CTLA-4 antibodyis tremelimumab.

In some embodiments, the anti-CTLA-4 antibody is administered at a doseranging from at least about 0.01 mg/kg to at least about 10 mg/kg bodyweight once about every 1, 2 or 3 weeks. In one embodiment, theanti-CTLA-4 antibody is administered at a dose of at least about 1 mg/kgbody weight once about every 3 weeks. In another embodiment, theanti-CTLA-4 antibody is administered at a dose of at least about 3 mg/kgbody weight once about every 3 weeks. In some embodiments, theanti-CTLA-4 antibody is administered for as long as clinical benefit isobserved or until unmanageable toxicity or disease progression occurs.

In some embodiments, the anti-PD-1 and anti-CTLA-4 antibodies areformulated for intravenous administration. In certain embodiments, theanti-PD-1 and anti-CTLA-4 antibodies are administered sequentially. Inone embodiment, the anti-PD-1 and anti-CTLA-4 antibodies areadministered within 30 minutes of each other. In an embodiment, theanti-PD-1 antibody is administered before the anti-CTLA-4 antibody. Inanother embodiment, the anti-CTLA-4 antibody is administered before theanti-PD-1 antibody. In certain embodiments, the anti-PD-1 antibody andthe anti-CTLA-4 antibody are administered concurrently in separatecompositions. In other embodiments, the anti-PD-1 antibody and theanti-CTLA-4 antibody are admixed as a single composition for concurrentadministration.

In one embodiment, the anti-PD-1 antibody is administered at asubtherapeutic dose. In another embodiment, the anti-CTLA-4 antibody isadministered at a subtherapeutic dose. In a further embodiment, theanti-PD-1 antibody and the anti-CTLA-4 antibody are each administered ata subtherapeutic dose.

In certain embodiments, the subject has a tumor that expresses PD-L1,PD-L2, or both. In embodiments, the subject exhibits progression-freesurvival of at least about one month, at least about 2 months, at leastabout 3 months, at least about 4 months, at least about 5 months, atleast about 6 months, at least about 7 months, at least about 8 months,at least about 9 months, at least about 10 months, at least about 11months, at least about one year, at least about eighteen months, atleast about two years, at least about three years, at least about fouryears, or at least about five years after the initial administration.

The present disclosure relates to a kit for treating a subject afflictedwith a cancer, the kit comprising: (a) a dosage ranging from about 0.1mg/kg to about 10 mg/kg of an anti-PD-1 antibody; (b) a dosage rangingfrom about 0.1 mg/kg to about 10 mg/kg of an anti-CTLA-4 antibody; and(c) instructions for using the anti-PD-1 antibody and the anti-CTLA-4antibody in any method disclosed herein.

In some embodiments, the subject has a microsatellite stable (MSS) tumoror a microsatellite instability-high (MSI-H) tumor. Certain embodimentsfurther comprise measuring the microsatellite status of a tumor prior toadministration of an anti-PD-1 antibody and/or an anti-CTLA-4 antibody.In embodiments, the tumor is a MSS tumor or a MSI-H tumor. In oneembodiment, the subject is afflicted with a colorectal cancer. Inanother embodiment, the subject is afflicted with colon cancer. In yetanother embodiment, the subject is afflicted with a rectal cancer.

Embodiments

E1. A method for treating a subject afflicted with a tumor derived froma colorectal cancer comprising administering to the subject atherapeutically effective amount of an antibody or an antigen-bindingportion thereof that binds specifically to Programmed Death-1 receptor(PD-1) and inhibits PD-1 activity (“anti-PD-1 antibody”), wherein thetumor exhibits a high degree of microsatellite instability (“MSI-H”).

E2. The method of embodiment E1, wherein the tumor is a colon cancer.

E3. The method of embodiment E1, wherein the tumor is a rectal cancer.

E4. The method of any one of embodiments E1 to E3, wherein the tumorexhibits one or more characteristics selected from the group consistingof: (a) the tumor comprises a germline alteration in at least two, atleast three, at least four, or at least five DNA mismatch repair genes(“MMR genes”); (b) the tumor comprises a germline alteration in at least30% of five or more MMR genes; (c) at least one protein encoded by DNAMMR genes is not detected in the tumor; and (d) any combination thereof.

E5. The method of embodiment E4, wherein the DNA MMR genes compriseMSH2, MLH1, MSH6, PMS2, PMS1, or any combination thereof.

E6. The method of embodiment E4, wherein the germline alternation in (a)or (b) is measured by a polymerase chain reaction.

E7. The method of embodiment E4, wherein the at least one proteinencoded by DNA MMR genes is detected by an immunohistochemistry.

E8. The method of any one of embodiments E1 to E7, wherein the subjectexhibits a progression-free survival of at least about one month, atleast about 2 months, at least about 3 months, at least about 4 months,at least about 5 months, at least about 6 months, at least about 7months, at least about 8 months, at least about 9 months, at least about10 months, at least about 11 months, or at least about 12 months afterthe administration of treatment.

E9. The method of any one of embodiments E1 to E8, wherein the subjectexhibits an overall survival of at least about one month, at least about2 months, at least about 3 months, at least about 4 months, at leastabout 5 months, at least about 6 months, at least about 7 months, atleast about 8 months, at least about 9 months, at least about 10 months,at least about 11 months, or at least about 12 months after theadministration of treatment.

E10. The method of any one of embodiments E1 to E9, wherein theadministering treats the cancer.

E11. The method of any one of embodiments E1 to E10, wherein theanti-PD-1 antibody cross-competes with nivolumab for binding to humanPD-1.

E12. The method of any one of embodiments E1 to E11, wherein theanti-PD-1 antibody binds to the same epitope as nivolumab.

E13. The method of any one of embodiments E1 to E12, wherein theanti-PD-1 antibody is a chimeric, humanized or human monoclonal antibodyor a portion thereof.

E14. The method of any one of embodiments E1 to E13, wherein theanti-PD-1 antibody comprises a heavy chain constant region which is of ahuman IgG1 or IgG4 isotype.

E15. The method of any one of embodiments E1 to E14, wherein theanti-PD-1 antibody is nivolumab.

E16. The method of any one of embodiments E1 to E14, wherein theanti-PD-1 antibody is pembrolizumab.

E17. The method of any one of embodiments E1 to E16, wherein theanti-PD-1 antibody is administered at a dose ranging from at least about0.1 mg/kg to at least about 10.0 mg/kg body weight once about every 1, 2or 3 weeks.

E18. The method of embodiment E17, wherein the anti-PD-1 antibody isadministered at a dose of about 3 mg/kg body weight once about every 3weeks.

E19. The method of any one of embodiments E1 to E18, further comprisingadministering a therapeutically effective amount of an antibody orantigen-binding portion thereof that specifically binds to CTLA-4 andinhibits CTLA-4 activity (“anti-CTLA-4 antibody”).

E20. The method of embodiment E19, wherein the anti-CTLA-4 antibodycross-competes with ipilimumab for binding to human CTLA-4.

E21. The method of embodiment E19 or E20, wherein the anti-CTLA-4antibody binds to the same epitope as ipilimumab.

E22. The method of any one of embodiments E19 to E21, wherein theanti-CTLA-4 antibody is a chimeric, humanized or human monoclonalantibody or a portion thereof.

E23. The method of any one of embodiments E19 to E22, wherein theanti-CTLA-4 antibody comprises a heavy chain constant region which is ofa human IgG1 or IgG2 isotype.

E24. The method of any one of embodiments E19 to E23, wherein theanti-CTLA-4 antibody is ipilimumab.

E25. The method of any one of embodiments E19 to E23, wherein theanti-CTLA-4 antibody is tremelimumab.

E26. The method of any one of embodiments E19 to E25, wherein theanti-CTLA-4 antibody is administered at a dose ranging from at leastabout 0.1 mg/kg to at least about 10.0 mg/kg body weight once aboutevery 1, 2 or 3 weeks.

E27. The method of embodiment E26, wherein the anti-CTLA-4 antibody isadministered at a dose of about 1 mg/kg body weight once about every 3weeks.

E28. The method of any one of embodiments E19 to E25, wherein theanti-PD-1 antibody is administered at a dose of about 1 mg/kg bodyweight and the anti-CTLA-4 antibody is administered at a dose of about 1mg/kg body weight.

E29. The method of any one of embodiments E19 to E25, wherein theanti-PD-1 antibody is administered at a dose of about 1 mg/kg bodyweight and the anti-CTLA-4 antibody is administered at a dose of about 3mg/kg body weight.

E30. The method of any one of embodiments E19 to E25, wherein theanti-PD-1 antibody is administered at a dose of about 3 mg/kg bodyweight and the anti-CTLA-4 antibody is administered at a dose of about 1mg/kg body weight.

E31. The method of any one of embodiments E19 to E25, wherein theanti-PD-1 antibody is administered at a dose of about 3 mg/kg bodyweight and the anti-CTLA-4 antibody is administered at a dose of about 3mg/kg body weight.

E32. The method of any one of embodiments E19 to E31, wherein theadministration of an anti-PD-1 antibody and anti-CTLA-4 antibody isfollowed by an anti-PD-1 antibody monotherapy.

E33. The method of embodiment E32, wherein the anti-PD-1 antibodymonotherapy comprises administering an anti-PD-1 antibody at a dose ofabout 1 mg/kg body weight.

E34. The method of embodiment E32, wherein the anti-PD-1 antibodymonotherapy comprises administering an anti-PD-1 antibody at a dose ofabout 3 mg/kg body weight.

E35. The method of embodiment E32, wherein the anti-PD-1 antibodymonotherapy comprises administering an anti-PD-1 antibody at a dose ofabout 4 mg/kg body weight.

E36. The method of any one of embodiments E19 to E35, wherein theanti-PD-1 antibody and anti-CTLA-4 antibody are administered once aboutevery 1, 2, 3, or 4 weeks.

E37. The method of any one of embodiments E32 to E35, wherein theanti-PD-1 antibody monotherapy is administered once about every 1, 2, 3,or 4 weeks.

E38. The method of any one of embodiments E1 to E37, wherein the subjectexhibits a complete response or partial response to treatment at about 4weeks, about 5 weeks, about 6 weeks, about 7 weeks, about 8 weeks, about9 weeks, about 10 weeks, about 11 weeks, about 12 weeks, about 18 weeks,about 24 weeks, about 30 weeks, or about 36 weeks following the initialadministration of treatment.

E39. The method of any one of embodiments E19 to E38, wherein theanti-PD-1 and anti-CTLA-4 antibodies are formulated for intravenousadministration.

E40. The method of any one of embodiments E19 to E39, wherein theanti-PD-1 and anti-CTLA-4 antibodies are administered sequentially.

E41. The method of any one of embodiments E19 to E40, wherein theanti-PD-1 and anti-CTLA-4 antibodies are administered within 30 minutesof each other.

E42. The method of any one of embodiments E19 to E41, wherein theanti-PD-1 antibody is administered before the anti-CTLA-4 antibody.

E43. The method of any one of embodiments E19 to E41, wherein theanti-CTLA-4 antibody is administered before the anti-PD-1 antibody.

E44. The method of any one of embodiments E1 to E39, wherein theanti-PD-1 antibody and the anti-CTLA-4 antibody are administeredconcurrently in separate compositions.

E45. The method of any one of embodiments E1 to E39, wherein theanti-PD-1 antibody and the anti-CTLA-4 antibody are admixed as a singlecomposition for concurrent administration.

E46. A kit for treating a patient afflicted with a tumor derived fromcolorectal cancer, wherein the tumor exhibits a high degree ofmicrosatellite instability (“MSI-H”), the kit comprising: (a) a dosageranging from 0.1 mg/kg to 10 mg/kg body weight of an anti-PD-1 antibody;(b) a dosage ranging from 0.1 mg/kg to 10 mg/kg body weight of ananti-CTLA-4 antibody; and (c) instructions for using the anti-PD-1antibody and the anti-CTLA-4 antibody in the method of any one ofembodiments E1 to E45.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a study schematic for microsatellite stable (MSS)colorectal cancer patients receiving 1 mg/kg of nivolumab in combinationwith 3 mg/kg of ipilimumab or 3 mg/kg of nivolumamb in combination with1 mg/kg ipilimumab.

FIG. 2 shows a study schematic for microsatellite instability-high(MSI-H) colorectal cancer patients receiving 3 mg/kg nivolumabmonotherapy or 3 mg/kg nivolumab in combination with 1 mg/kg ipilimumab.

FIG. 3 shows the investigator-assessed objective responses of MSI-Hcolorectal cancer patients receiving 3 mg/kg nivolumab monotherapy.

FIG. 4 shows the investigator-assessed objective responses of MSI-Hcolorectal cancer patients receiving 3 mg/kg nivolumab in combinationwith 1 mg/kg ipilimumab.

FIGS. 5A-5B show the best reduction in target lesion size for MSI-Hcolorectal cancer patients receiving 3 mg/kg nivolumab monotherapy (FIG.5A) compared to MSI-H colorectal cancer patients receiving 3 mg/kgnivolumab in combination with 1 mg/kg ipilimumab (FIG. 5B).

FIG. 6 shows the investigator-assessed progression-free survival ofMSI-H colorectal cancer patients receiving 3 mg/kg nivolumab monotherapyor 3 mg/kg nivolumab in combination with 1 mg/kg ipilimumab.

FIG. 7 shows the overall survival of MSI-H colorectal cancer patientsreceiving 3 mg/kg nivolumab monotherapy or 3 mg/kg nivolumab incombination with 1 mg/kg ipilimumab.

FIG. 8 shows a study schematic for DNA mismatchrepair-deficient/microsatellite instability-high (dMMR/MSI-H) metastaticcolorectal cancer patients receiving 3 mg/kg nivolumab monotherapy or 3mg/kg nivolumab+1 mg/kg ipilimumab combination therapy.

FIG. 9 shows the best reduction from baseline in target lesion size fordMMR/MSI-H metastatic colorectal cancer patients receiving nivolumabmonotherapy in an extended clinical case study.

FIG. 10 shows the best reduction from baseline in target lesion size fordMMR/MSI-H metastatic colorectal cancer patients receivingnivolumab+ipilimumab combination therapy.

FIG. 11 shows the change in tumor burden over time for dMMR/MSI-Hmetastatic colorectal cancer patients receiving nivolumab+ipilimumabcombination therapy.

FIGS. 12A-12B show the progression-free survival (PFS) per investigatorassessment (FIG. 12A) and overall survival (OS) per investigatorassessment (FIG. 12B) for dMMR/MSI-H metastatic colorectal cancerpatients receiving nivolumab+ipilimumab combination therapy.

DETAILED DESCRIPTION OF THE DISCLOSURE

This disclosure relates to methods for treating a colorectal cancer in asubject comprising administering to the subject an anti-ProgrammedDeath-1 (PD-1) antibody. In some embodiments, the colorectal cancerexhibits a high degree of microsatellite instability (MSI-H). In someembodiments, the method further comprises administering ananti-Cytotoxic T-Lymphocyte Antigen-4 (CTLA-4) antibody. In someembodiments, the colorectal cancer is rectal cancer, colon cancer, orany combination thereof.

Terms

In order that the present disclosure may be more readily understood,certain terms are first defined. As used in this application, except asotherwise expressly provided herein, each of the following terms shallhave the meaning set forth below. Additional definitions are set forththroughout the application.

The term “and/or” where used herein is to be taken as specificdisclosure of each of the two specified features or components with orwithout the other. Thus, the term “and/or” as used in a phrase such as“A and/or B” herein is intended to include “A and B,” “A or B,” “A”(alone), and “B” (alone). Likewise, the term “and/or” as used in aphrase such as “A, B, and/or C” is intended to encompass each of thefollowing aspects: A, B, and C; A, B, or C; A or C; A or B; B or C; Aand C; A and B; B and C; A (alone); B (alone); and C (alone).

It is understood that wherever aspects are described herein with thelanguage “comprising,” otherwise analogous aspects described in terms of“consisting of” and/or “consisting essentially of” are also provided.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this disclosure is related. For example, the ConciseDictionary of Biomedicine and Molecular Biology, Juo, Pei-Show, 2nd ed.,2002, CRC Press; The Dictionary of Cell and Molecular Biology, 3rd ed.,1999, Academic Press; and the Oxford Dictionary Of Biochemistry AndMolecular Biology, Revised, 2000, Oxford University Press, provide oneof skill with a general dictionary of many of the terms used in thisdisclosure.

Units, prefixes, and symbols are denoted in their Systeme Internationalde Unites (SI) accepted form. Numeric ranges are inclusive of thenumbers defining the range. The headings provided herein are notlimitations of the various aspects of the disclosure, which can be hadby reference to the specification as a whole. Accordingly, the termsdefined immediately below are more fully defined by reference to thespecification in its entirety.

“Administering” refers to the physical introduction of a therapeuticagent to a subject, using any of the various methods and deliverysystems known to those skilled in the art. Exemplary routes ofadministration for the anti-PD-1 antibody include intravenous,intramuscular, subcutaneous, intraperitoneal, spinal or other parenteralroutes of administration, for example by injection or infusion. Thephrase “parenteral administration” as used herein means modes ofadministration other than enteral and topical administration, usually byinjection, and includes, without limitation, intravenous, intramuscular,intraarterial, intrathecal, intralymphatic, intralesional,intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal,transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular,subarachnoid, intraspinal, epidural and intrasternal injection andinfusion, as well as in vivo electroporation. A therapeutic agent may beadministered via a non-parenteral route, or orally. Other non-parenteralroutes include a topical, epidermal or mucosal route of administration,for example, intranasally, vaginally, rectally, sublingually ortopically. Administering can also be performed, for example, once, aplurality of times, and/or over one or more extended periods.

An “adverse event” (AE) as used herein is any unfavorable and generallyunintended or undesirable sign (including an abnormal laboratoryfinding), symptom, or disease associated with the use of a medicaltreatment. A medical treatment may have one or more associated AEs andeach AE may have the same or different level of severity. Reference tomethods capable of “altering adverse events” means a treatment regimethat decreases the incidence and/or severity of one or more AEsassociated with the use of a different treatment regime.

An “antibody” (Ab) shall include, without limitation, a glycoproteinimmunoglobulin which binds specifically to an antigen and comprises atleast two heavy (H) chains and two light (L) chains interconnected bydisulfide bonds, or an antigen-binding portion thereof. Each H chaincomprises a heavy chain variable region (abbreviated herein as V_(H))and a heavy chain constant region. The heavy chain constant regioncomprises at least three constant domains, C_(H1), C_(H2) and C_(H3).Each light chain comprises a light chain variable region (abbreviatedherein as V_(L)) and a light chain constant region. The light chainconstant region comprises one constant domain, C_(L). The V_(H) andV_(L) regions can be further subdivided into regions ofhypervariability, termed complementarity determining regions (CDRs),interspersed with regions that are more conserved, termed frameworkregions (FRs). Each V_(H) and V_(L) comprises three CDRs and four FRs,arranged from amino-terminus to carboxy-terminus in the following order:FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. The variable regions of theheavy and light chains contain a binding domain that interacts with anantigen. The constant regions of the antibodies may mediate the bindingof the immunoglobulin to host tissues or factors, including variouscells of the immune system (e.g., effector cells) and the firstcomponent (C1q) of the classical complement system.

An immunoglobulin may derive from any of the commonly known isotypes,including but not limited to IgA, secretory IgA, IgG and IgM. IgGsubclasses are also well known to those in the art and include but arenot limited to human IgG1, IgG2, IgG3 and IgG4. “Isotype” refers to theantibody class or subclass (e.g., IgM or IgG1) that is encoded by theheavy chain constant region genes. The term “antibody” includes, by wayof example, both naturally occurring and non-naturally occurringantibodies; monoclonal and polyclonal antibodies; chimeric and humanizedantibodies; human or non-human antibodies; wholly synthetic antibodies;and single chain antibodies. A non-human antibody may be humanized byrecombinant methods to reduce its immunogenicity in man. Where notexpressly stated, and unless the context indicates otherwise, the term“antibody” also includes an antigen-binding fragment or anantigen-binding portion of any of the aforementioned immunoglobulins,and includes a monovalent and a divalent fragment or portion, and asingle chain antibody.

An “isolated antibody” refers to an antibody that is substantially freeof other antibodies having different antigenic specificities (e.g., anisolated antibody that binds specifically to PD-1 is substantially freeof antibodies that bind specifically to antigens other than PD-1). Anisolated antibody that binds specifically to PD-1 may, however, havecross-reactivity to other antigens, such as PD-1 molecules fromdifferent species. Moreover, an isolated antibody may be substantiallyfree of other cellular material and/or chemicals.

The term “monoclonal antibody” (“mAb”) refers to a non-naturallyoccurring preparation of antibody molecules of single molecularcomposition, i.e., antibody molecules whose primary sequences areessentially identical, and which exhibits a single binding specificityand affinity for a particular epitope. A monoclonal antibody is anexample of an isolated antibody. Monoclonal antibodies may be producedby hybridoma, recombinant, transgenic or other techniques known to thoseskilled in the art.

A “human” antibody (HuMAb) refers to an antibody having variable regionsin which both the framework and CDRs are derived from human germlineimmunoglobulin sequences. Furthermore, if the antibody contains aconstant region, the constant region also is derived from human germlineimmunoglobulin sequences. The human antibodies of the disclosure mayinclude amino acid residues not encoded by human germline immunoglobulinsequences (e.g., mutations introduced by random or site-specificmutagenesis in vitro or by somatic mutation in vivo). However, the term“human antibody,” as used herein, is not intended to include antibodiesin which CDR sequences derived from the germline of another mammalianspecies, such as a mouse, have been grafted onto human frameworksequences. The terms “human” antibodies and “fully human” antibodies andare used synonymously.

A “humanized antibody” refers to an antibody in which some, most or allof the amino acids outside the CDRs of a non-human antibody are replacedwith corresponding amino acids derived from human immunoglobulins. Inone embodiment of a humanized form of an antibody, some, most or all ofthe amino acids outside the CDRs have been replaced with amino acidsfrom human immunoglobulins, whereas some, most or all amino acids withinone or more CDRs are unchanged. Small additions, deletions, insertions,substitutions or modifications of amino acids are permissible as long asthey do not abrogate the ability of the antibody to bind to a particularantigen. A “humanized” antibody retains an antigenic specificity similarto that of the original antibody. In some embodiments, the CDRs of ahumanized antibody contain CDRs from a non-human, mammalian antibody. Inother embodiments, the CDRs of a humanized antibody contain CDRs from anengineered, synthetic antibody.

A “chimeric antibody” refers to an antibody in which the variableregions are derived from one species and the constant regions arederived from another species, such as an antibody in which the variableregions are derived from a mouse antibody and the constant regions arederived from a human antibody.

An “anti-antigen” antibody refers to an antibody that binds specificallyto the antigen. For example, an anti-PD-1 antibody binds specifically toPD-1 and an anti-CTLA-4 antibody binds specifically to CTLA-4.

An “antigen-binding portion” of an antibody (also called an“antigen-binding fragment”) refers to one or more fragments of anantibody that retain the ability to bind specifically to the antigenbound by the whole antibody.

A “cancer” refers a broad group of various diseases characterized by theuncontrolled growth of abnormal cells in the body. A “cancer” or “cancertissue” can include a tumor. Unregulated cell division and growthresults in the formation of malignant tumors that invade neighboringtissues and may also metastasize to distant parts of the body throughthe lymphatic system or bloodstream. Following metastasis, the distaltumors can be said to be “derived from” the original, pre-metastasistumor. For example, a “tumor derived from” a colorectal cancer refers toa tumor that is the result of a metastasized colorectal cancer. Becausethe distal tumor is derived from the pre-metastasis tumor, the “derivedfrom” tumor can also comprise the pre-metastasis tumor, e.g., a tumorderived from a colorectal cancer can comprise a colorectal cancer.

“Cytotoxic T-Lymphocyte Antigen-4” (CTLA-4) refers to animmunoinhibitory receptor belonging to the CD28 family. CTLA-4 isexpressed exclusively on T cells in vivo, and binds to two ligands, CD80and CD86 (also called B7-1 and B7-2, respectively). The term “CTLA-4” asused herein includes human CTLA-4 (hCTLA-4), variants, isoforms, andspecies homologs of hCTLA-4, and analogs having at least one commonepitope with hCTLA-4. The complete hCTLA-4 sequence can be found underGenBank Accession No. AAB59385.

The term “immunotherapy” refers to the treatment of a subject afflictedwith, or at risk of contracting or suffering a recurrence of, a diseaseby a method comprising inducing, enhancing, suppressing or otherwisemodifying an immune response.

“Treatment” or “therapy” of a subject refers to any type of interventionor process performed on, or the administration of an active agent to,the subject with the objective of reversing, alleviating, ameliorating,inhibiting, slowing down or preventing the onset, progression,development, severity or recurrence of a symptom, complication orcondition, or biochemical indicia associated with a disease.

“PD-L1 positive” or “PD-L2 positive” as used herein can beinterchangeably used with “PD-L1 and/or PD-L2 expression of at leastabout 1%.” In one embodiment, the PD-L1 and/or PD-L2 expression can beused by any methods known in the art. In another embodiment, the PD-L1and/or PD-L2 expression is measured by an automated in situhybridization (IHC). A PD-L1 and/or PD-L2 positive tumor can thus haveat least about 1%, at least about 2%, at least about 5%, at least about10%, or at least about 20%, at least about 25%, at least about 30%, atleast about 40%, at least about 50%, at least about 60%, at least about70%, at least about 75%, at least about 80%, at least about 85%, atleast about 90%, at least about 95%, or about 100% of tumor cellsexpressing PD-L1 and/or PD-L2 as measured by an automated IHC. Incertain embodiments, “PD-L1 positive” means that there are at least 100cells that express PD-L1 on the surface of the cells. In otherembodiments, “PD-L2 positive” means that there are at least 100 cellsthat express PD-L2 on the surface of the cells.

“Programmed Death-1 (PD-1)” refers to an immunoinhibitory receptorbelonging to the CD28 family. PD-1 is expressed predominantly onpreviously activated T cells in vivo, and binds to two ligands, PD-L1and PD-L2. The term “PD-1” as used herein includes human PD-1 (hPD-1),variants, isoforms, and species homologs of hPD-1, and analogs having atleast one common epitope with hPD-1. The complete hPD-1 sequence can befound under GenBank Accession No. U64863.

“Programmed Death Ligand-1” (PD-L1) is one of two cell surfaceglycoprotein ligands for PD-1 (the other being PD-L2) that down regulateT cell activation and cytokine secretion upon binding to PD-1. The term“PD-L1” as used herein includes human PD-L1 (hPD-L1), variants,isoforms, and species homologs of hPD-L1, and analogs having at leastone common epitope with hPD-L1. The complete hPD-L1 sequence can befound under GenBank Accession No. Q9NZQ7.

A “subject” includes any human or non-human animal. The term “non-humananimal” includes, but is not limited to, vertebrates such as non-humanprimates, sheep, dogs, and rodents such as mice, rats and guinea pigs.In some embodiments, the subject is a human. The terms, “subject” and“patient” are used interchangeably herein.

A “therapeutically effective amount” or “therapeutically effectivedosage” of a drug or therapeutic agent is any amount of the drug that,when used alone or in combination with another therapeutic agent,protects a subject against the onset of a disease or promotes diseaseregression evidenced by a decrease in severity of disease symptoms, anincrease in frequency and duration of disease symptom-free periods, or aprevention of impairment or disability due to the disease affliction.The ability of a therapeutic agent to promote disease regression can beevaluated using a variety of methods known to the skilled practitioner,such as in human subjects during clinical trials, in animal modelsystems predictive of efficacy in humans, or by assaying the activity ofthe agent in in vitro assays.

As used herein, “subtherapeutic dose” means a dose of a therapeuticcompound (e.g., an antibody) that is lower than the usual or typicaldose of the therapeutic compound when administered alone for thetreatment of a hyperproliferative disease (e.g., cancer).

By way of example, an “anti-cancer agent” promotes cancer regression ina subject. In some embodiments, a therapeutically effective amount ofthe drug promotes cancer regression to the point of eliminating thecancer. “Promoting cancer regression” means that administering aneffective amount of the drug, alone or in combination with ananti-cancer agent, results in a reduction in tumor growth or size,necrosis of the tumor, a decrease in severity of at least one diseasesymptom, an increase in frequency and duration of disease symptom-freeperiods, or a prevention of impairment or disability due to the diseaseaffliction. In addition, the terms “effective” and “effectiveness” withregard to a treatment includes both pharmacological effectiveness andphysiological safety. Pharmacological effectiveness refers to theability of the drug to promote cancer regression in the patient.Physiological safety refers to the level of toxicity, or other adversephysiological effects at the cellular, organ and/or organism level(adverse effects) resulting from administration of the drug.

By way of example for the treatment of tumors, a therapeuticallyeffective amount of an anti-cancer agent inhibits cell growth or tumorgrowth by at least about 10%, by at least about 20%, by at least about30%, by at least about 40%, by at least about 50%, by at least about60%, by at least about 70%, by at least about 80%, by at least about90%, or about 100% relative to untreated subjects.

In other embodiments of the disclosure, tumor regression can be observedand continue for a period of at least about 20 days, at least about 30days, at least about 40 days, at least about 50 days, or at least about60 days. Notwithstanding these ultimate measurements of therapeuticeffectiveness, evaluation of immunotherapeutic drugs must also makeallowance for “immune-related” response patterns.

An “immune-related” response pattern refers to a clinical responsepattern often observed in cancer patients treated with immunotherapeuticagents that produce antitumor effects by inducing cancer-specific immuneresponses or by modifying native immune processes. This response patternis characterized by a beneficial therapeutic effect that follows aninitial increase in tumor burden or the appearance of new lesions, whichin the evaluation of traditional chemotherapeutic agents would beclassified as disease progression and would be synonymous with drugfailure. Accordingly, proper evaluation of immunotherapeutic agents mayrequire long-term monitoring of the effects of these agents on thetarget disease.

A therapeutically effective amount of a drug includes a“prophylactically effective amount,” which is any amount of the drugthat, when administered alone or in combination with an anti-canceragent to a subject at risk of developing a cancer (e.g., a subjecthaving a pre-malignant condition) or of suffering a recurrence ofcancer, inhibits the development or recurrence of the cancer. In someembodiments, the prophylactically effective amount prevents thedevelopment or recurrence of the cancer entirely. “Inhibiting” thedevelopment or recurrence of a cancer means either lessening thelikelihood of the cancer's development or recurrence, or preventing thedevelopment or recurrence of the cancer entirely.

The term “weight-based dose” as referred to herein means that a dosethat is administered to a patient is calculated based on the weight ofthe patient. For example, when a patient with 60 kg body weight requires3 mg/kg of an anti-PD-1 antibody, one can calculate and use theappropriate amount of the anti-PD-1 antibody (i.e., 180 mg) foradministration.

The use of the term “fixed dose” with regard to a method of thedisclosure means that two or more different antibodies in a singlecomposition (e.g., anti-PD-1 antibody and anti-CTLA-4 antibody) arepresent in the composition in particular (fixed) ratios with each other.In some embodiments, the fixed dose is based on the weight (e.g., mg) ofthe antibodies. In certain embodiments, the fixed dose is based on theconcentration (e.g., mg/ml) of the antibodies. In some embodiments, theratio is at least about 1:1, about 1:2, about 1:3, about 1:4, about 1:5,about 1:6, about 1:7, about 1:8, about 1:9, about 1:10, about 1:15,about 1:20, about 1:30, about 1:40, about 1:50, about 1:60, about 1:70,about 1:80, about 1:90, about 1:100, about 1:120, about 1:140, about1:160, about 1:180, about 1:200, about 200:1, about 180:1, about 160:1,about 140:1, about 120:1, about 100:1, about 90:1, about 80:1, about70:1, about 60:1, about 50:1, about 40:1, about 30:1, about 20:1, about15:1, about 10:1, about 9:1, about 8:1, about 7:1, about 6:1, about 5:1,about 4:1, about 3:1, or about 2:1 mg first antibody (e.g., anti-PD-1antibody) to mg second antibody (e.g., anti-CTLA-4 antibody). Forexample, the 3:1 ratio of an anti-PD-1 antibody and an anti-CTLA-4antibody can mean that a vial can contain about 240 mg of the anti-PD-1antibody and 80 mg of the anti-CTLA-4 antibody or about 3 mg/ml of theanti-PD-1 antibody and 1 mg/ml of the anti-CTLA-4 antibody.

The use of the term “flat dose” with regard to the methods and dosagesof the disclosure means a dose that is administered to a patient withoutregard for the weight or body surface area (B S A) of the patient. Theflat dose is therefore not provided as a mg/kg dose, but rather as anabsolute amount of the agent (e.g., the anti-CTLA-4 antibody and/oranti-PD-1 antibody). For example, a 60 kg person and a 100 kg personwould receive the same dose of an antibody (e.g., 240 mg of an anti-PD1antibody).

The use of the alternative (e.g., “or”) should be understood to meaneither one, both, or any combination thereof of the alternatives. Asused herein, the indefinite articles “a” or “an” should be understood torefer to “one or more” of any recited or enumerated component.

The terms “about” or “comprising essentially of” refer to a value orcomposition that is within an acceptable error range for the particularvalue or composition as determined by one of ordinary skill in the art,which will depend in part on how the value or composition is measured ordetermined, i.e., the limitations of the measurement system. Forexample, “about” or “comprising essentially of” can mean within 1 ormore than 1 standard deviation per the practice in the art.Alternatively, “about” or “comprising essentially of” can mean a rangeof up to 20%. Furthermore, particularly with respect to biologicalsystems or processes, the terms can mean up to an order of magnitude orup to 5-fold of a value. When particular values or compositions areprovided in the application and claims, unless otherwise stated, themeaning of “about” or “comprising essentially of” should be assumed tobe within an acceptable error range for that particular value orcomposition.

The terms “once about every week,” “once about every two weeks,” or anyother similar dosing interval terms as used herein mean approximatenumbers. “Once about every week” can include every seven days±one day,i.e., every six days to every eight days. “Once about every two weeks”can include every fourteen days±three days, i.e., every eleven days toevery seventeen days. Similar approximations apply, for example, to onceabout every three weeks, once about every four weeks, once about everyfive weeks, once about every six weeks and once about every twelveweeks. In some embodiments, a dosing interval of once about every sixweeks or once about every twelve weeks means that the first dose can beadministered any day in the first week, and then the next dose can beadministered any day in the sixth or twelfth week, respectively. Inother embodiments, a dosing interval of once about every six weeks oronce about every twelve weeks means that the first dose is administeredon a particular day of the first week (e.g., Monday) and then the nextdose is administered on the same day of the sixth or twelfth weeks(i.e., Monday), respectively.

As described herein, any concentration range, percentage range, ratiorange or integer range is to be understood to include the value of anyinteger within the recited range and, when appropriate, fractionsthereof (such as one tenth and one hundredth of an integer), unlessotherwise indicated.

Various aspects of the disclosure are described in further detail in thefollowing subsections.

TABLE 1 List of Abbreviations Term Definition AEs adverse events BMSBristol-Myers Squibb CRC colorectal cancer CI confidence interval CRcomplete remission DOR duration of response Kg kilogram mAB monoclonalantibody Mg milligram N number of subjects or observations NE notevaluable ORR overall response rate OS overall survival PD progressivedisease PD-1 programmed death-1 PD-L1 programmed death-ligand 1 PD-L2programmed death-ligand 2 PFS progression-free survival PR partialremission SAE serious adverse event SD stable disease SOP StandardOperating Procedures Subj subject

Methods of the Disclosure

The present disclosure is directed to a method for treating a subjectafflicted with a tumor derived from a colorectal cancer comprisingadministering to the subject a therapeutically effective amount of anantibody or an antigen-binding portion thereof that binds specificallyto a Programmed Death-1 receptor (PD-1) and inhibits PD-1 activity(“anti-PD-1 antibody”) or an antibody or an antigen-binding portionthereof that binds specifically to a Programmed Death-Ligand 1 (PD-L1)and inhibits PD-L1 activity (“anti-PD-L1 antibody”).

In some embodiments, the therapy treats a tumor derived from a cancer,which is colorectal cancer. In some embodiments, the colorectal canceris colon cancer. In other embodiments, the colorectal cancer is rectalcancer. In certain embodiments, the colorectal cancer has microsatelliteinstability (MSI) (See Pawlik et al., Dis. Markers 20(4-5): 199-206(2004)). In other embodiments, the colorectal cancer has highmicrosatellite instability (MSI-H).

Colon cancer presents in five stages: Stage 0 (Carcinoma in Situ), StageI, Stage II, Stage III and Stage IV. Six types of standard treatment areused for colon cancer: 1) surgery, including a local excision, resectionof the colon with anastomosis, or resection of the colon with colostomy;2) radiofrequency ablation; 3) cryosurgery; 4) chemotherapy; 5)radiation therapy; and 6) targeted therapies, including monoclonalantibodies and angiogenesis inhibitors. In some embodiments, thecombination therapy of the disclosure treats a colon cancer along with astandard of care therapy.

Rectal cancer presents in five stages: Stage 0 (Carcinoma in Situ),Stage I, Stage II, Stage III and Stage IV. Six types of standardtreatment are used for rectal cancer: 1) Surgery, including polypectomy,local excision, resection, radiofrequency ablation, cryosurgery, andpelvic exenteration; 2) radiation therapy; 3) chemotherapy; and 4)targeted therapy, including monoclonal antibody therapy. In someembodiments, the methods of the disclosure treats a rectal cancer alongwith a standard of care therapy.

In colorectal cancer, MSI-H is associated with increases in immuneinfiltration and expression of immune checkpoint regulators. Therefore,the methods of the disclosure include treatment of a colorectal cancerexhibiting a high degree of microsatellite instability (MSI-H). In someembodiments, the anti-PD-1 antibody is administered in combination witha therapeutically effective amount of an antibody or an antigen-bindingportion thereof that binds specifically to CTLA-4 (“anti-CTLA-4antibody”).

Microsatellite instability is the condition of genetic hypermutabilitythat results from impaired DNA mismatch repair (MMR). The presence ofMSI represents phenotypic evidence that MMR is not functioning normally.In most cases, the genetic basis for instability in MSI tumors is aninherited germline alteration in any one of the five human MMR genes:MSH2, MLH1, MSH6, PMS2, and PMS1. In certain embodiments, the subjectreceiving tumor treatment has a high degree of microsatelliteinstability (MSI-H) and has at least one mutation in genes MSH2, MLH1,MSH6, PMS2, or PMS1. In other embodiments, subjects receiving tumortreatment within a control group have no microsatellite instability (MSSor MSI stable) and has no mutation in genes MSH2, MLH1, MSH6, PMS2, andPMS1.

The present disclosure is also directed to methods of treating a tumor,e.g., tumor in colon, comprising identifying a subject responsive to ananti-PD-1 antibody administered alone or in combination with ananti-CTLA-4 antibody, wherein the subject has a MSI-H tumor. In someembodiments, the disclosure provides a method of treating a tumor, e.g.,a tumor in colon, comprising (i) identifying a subject who has a tumorthat is a MSI-H tumor and (ii) administering an effective amount of ananti-PD-1 antibody alone or in combination with an effective amount ofan anti-CTLA-4 antibody to the subject. As used herein, MSI-H tumorsmean tumors having greater than at least about 30% of unstable MSIbiomarkers. In some embodiments, a colorectal cancer is MSI-H when agermline alteration is detected in at least two, at least three, atleast four, or at least five MMR genes. In other embodiments, acolorectal cancer is MSI-H when a germline alteration is detected in atleast 30% of five or more MMR genes. In some embodiments, a germlinealternation in MMR genes is measured by a polymerase chain reaction. Inother embodiments, a colorectal cancer is MSI-H when at least oneprotein encoded by DNA MMR genes is not detected in the tumor. In someembodiments, the at least one protein encoded by DNA MMR genes isdetected by an immunohistochemistry. In certain embodiments, the presentdisclosure is directed to a method of treating a cancer comprising 1)identifying the microsatellite status of a tumor and 2) administering atherapy to the subject based on the microsatellite status. In someembodiments, the subject has MSI-H.

In some embodiments, the colorectal cancer is rectal cancer, coloncancer, or any combination thereof. In certain embodiments, the subjecthas received one, two, three, four, five or more prior cancertreatments. In other embodiments, the subject is treatment-naïve. Insome embodiments, the subject has progressed on other cancer treatments.In embodiments, the cancer has reoccurred. In some embodiments, thecancer is metastatic. In other embodiments, the cancer is notmetastatic.

In some embodiments, the colorectal cancer is histologically confirmed.In certain embodiments, the colorectal cancer is metastatic orrecurrent. In embodiments, the subject has had progression during,after, or been intolerant following the last administration of standardtherapies. In certain embodiments, the subject has microsatelliteinstability. In other embodiments, the colorectal cancer has a highdegree of microsatellite instability (MSI-H).

In other embodiments, the colorectal cancer that has MSI-H expressesPD-L1. In yet other embodiments, the colorectal cancer that has MSI-Hexpresses at least 1%, at least 2%, at least 3%, at least 4%, at least5%, at least 10%, at least 20%, at least 30%, at least 40%, or at least50% membrane PD-L1.

In some embodiments, the present methods comprise administering aneffective amount of an anti-PD-1 antibody. In other embodiments, thepresent methods comprise administering an effective amount of ananti-PD-1 antibody and an effective amount of an anti-CTLA-4 antibody.An effective amount of an anti-PD-1 antibody and/or an anti-CTLA-4antibody can be a flat dose or a weight based dose.

In embodiments, the disclosure includes a method of treating a subjectafflicted with a tumor derived from colorectal cancer comprisingadministering an anti-PD-1 antagonist in combination with an anti-CTLA-4antibody to treat cancer. An “anti-PD-1 antagonist” as referred hereinincludes any molecule that inhibits interaction between PD-1 (receptor)and PD-L1 (ligand) such that the signal pathway of PD-1/PD-L1 isblocked. In other embodiments, an anti-PD-1 antagonist is a PD-1-Fcfusion protein. In certain embodiments, an anti-PD-1 antagonist includesan anti-PD-1 fusion protein, an antisense molecule, a small molecule, aribozyme, or a nanobody that inhibits or prevents interaction betweenPD-1 and PD-L1.

In certain embodiments, the therapy of the present disclosure (e.g.,administration of an anti-PD-1 antibody alone or in combination with ananti-CTLA-4 antibody) effectively increases the duration of survival ofthe subject. For example, the duration of survival of the subject isincreased by at least about 1 month, at least about 2 months, at leastabout 3 months, at least about 4 months, at least about 5 months, atleast about 6 months, at least about 7 months, at least about 8 months,at least about 9 months, at least about 10 months, at least about 11months or at least about 1 year or more.

In certain embodiments, the therapy of the present disclosureeffectively increases the duration of progression-free survival of thesubject. For example, the progression free survival of the subject isincreased by at least about 1 month, at least about 2 months, at leastabout 3 months, at least about 4 months, at least about 5 months, atleast about 6 months, at least about 7 months, at least about 8 months,at least about 9 months, at least about 10 months, at least about 11months or at least about 1 year. In certain embodiments, the therapy ofthe present disclosure effectively increases the response rate in agroup of subjects. For example, the response rate in a group of subjectsis increased by at least about 2%, at least about 3%, at least about 4%,at least about 5%, at least about 10%, at least about 15%, at leastabout 20%, at least about 25%, at least about 30%, at last about 35%, atleast about 40%, at least about 45%, at least about 50%, at least about55%, at least about 60%, at least about 70%, at least about 75%, atleast about 80%, at least about 85%, at least about 90%, at least about95%, at least about 99% or about 100%.

In some embodiments, the anti-PD-1 antibody is formulated forintravenous administration. In some embodiments, the anti-PD-1 andanti-CTLA-4 antibodies are formulated for intravenous administration. Incertain embodiments, the anti-PD-1 and anti-CTLA-4 antibodies areadministered sequentially. In embodiments, the anti-PD-1 and anti-CTLA-4antibodies are administered within 30 minutes of each other. In oneembodiment, the anti-PD-1 antibody or antigen-binding portion thereof isadministered before the anti-CTLA-4 antibody or antigen-binding portionthereof. In another embodiment, the anti-CTLA-4 antibody orantigen-binding portion thereof is administered before the anti-PD-1antibody or antigen-binding portion thereof. In another embodiment, theanti-PD-1 antibody or antigen-binding portion thereof and theanti-CTLA-4 antibody or antigen-binding portion thereof are administeredconcurrently in separate compositions. In a further embodiment, theanti-PD-1 antibody or antigen-binding portion thereof and theanti-CTLA-4 antibody or antigen-binding portion thereof are admixed as asingle composition for concurrent administration.

In some embodiments, the anti-PD-1 antibody and anti-CTLA-4 antibody areadministered in a fixed dose.

In some embodiments, the cancer is microsatellite stable (MSS) (or “MSIstable”) and therefore has no microsatellite instability. In otherembodiments, the cancer has a high degree of microsatellite instability(MSI-H).

Anti-PD-1 Antibodies and Anti-PD-L1 Antibodies

The therapy of the present disclosure can utilize an anti-PD-1 antibodyor an antigen-binding portion thereof. PD-1 is a key immune checkpointreceptor expressed by activated T and B cells and mediatesimmunosuppression. PD-1 is a member of the CD28 family of receptors,which includes CD28, CTLA-4, ICOS, PD-1, and BTLA. Two cell surfaceglycoprotein ligands for PD-1 have been identified, Programmed DeathLigand-1 (PD-L1) and Programmed Death Ligand-2 (PD-L2), that areexpressed on antigen-presenting cells as well as many human cancers andhave been shown to down regulate T cell activation and cytokinesecretion upon binding to PD-1. Inhibition of the PD-1/PD-L1 interactionmediates potent antitumor activity in preclinical models.

Anti-PD-1 antibodies suitable for use in the disclosed methods areantibodies that bind to PD-1 with high specificity and affinity, blockthe binding of PD-L1, and inhibit the immunosuppressive effect of thePD-1 signaling pathway. In any of the therapeutic methods disclosedherein, an anti-PD-1 or anti-PD-L1 “antibody” includes anantigen-binding portion that binds to the PD-1 or PD-L1 receptor,respectively, and exhibits the functional properties similar to those ofwhole antibodies in inhibiting ligand binding and upregulating theimmune system. In certain embodiments, the anti-PD-1 antibody orantigen-binding portion thereof cross-competes with nivolumab forbinding to human PD-1. In other embodiments, the anti-PD-L1 antibody orantigen-binding fragment thereof competes for binding with BMS-936559,MPDL3280A, MEDI4736, or MSB0010718C for binding to human PD-L1.

In other embodiments, the anti-PD-1 antibody or anti-PD-L1 antibody, orantigen-binding portions thereof is a chimeric, humanized, or humanmonoclonal antibody or a portion thereof. In certain embodiments fortreating a human subject, the antibody is a humanized antibody. In otherembodiments for treating a human subject, the antibody is a humanantibody of an IgG1, IgG2, IgG3, or IgG4 isotype can be used.

In certain embodiments, the anti-PD-1 antibody, or anti-PD-L1 antibody,or antigen-binding portions thereof comprises a heavy chain constantregion which is of a human IgG1 or IgG4 isotype. In certain otherembodiments, the sequence of the IgG4 heavy chain constant region of theanti-PD-1 antibody or anti-PD-L1 antibody, or antigen-binding portionsthereof, contains an S228P mutation which replaces a serine residue inthe hinge region with the proline residue normally found at thecorresponding position in IgG1 isotype antibodies. This mutation, whichis present in nivolumab, prevents Fab arm exchange with endogenous IgG4antibodies, while retaining the low affinity for activating Fc receptorsassociated with wild-type IgG4 antibodies (Wang et al., In vitrocharacterization of the anti-PD-1 antibody nivolumab, BMS-936558, and invivo toxicology in non-human primates, Cancer Imm Res, 2(9):846-56(2014)). In yet other embodiments, the antibody comprises a light chainconstant region which is a human kappa or lambda constant region. Inother embodiments, the anti-PD-1 antibody, or anti-PD-L1 antibody, orantigen-binding portions thereof is a monoclonal antibody or anantigen-binding portion thereof.

Human monoclonal antibodies that bind specifically to PD-1 with highaffinity have been disclosed in U.S. Pat. No. 8,008,449. Other anti-PD-1monoclonal antibodies have been described in, for example, U.S. Pat.Nos. 6,808,710, 7,488,802, 8,168,757 and 8,354,509, and PCT PublicationNo. WO 2012/145493. Each of the anti-PD-1 human monoclonal antibodiesdisclosed in U.S. Pat. No. 8,008,449 has been demonstrated to exhibitone or more of the following characteristics: (a) binds to human PD-1with a K_(D) of 1×10⁻⁷ M or less, as determined by surface plasmonresonance using a Biacore biosensor system; (b) does not substantiallybind to human CD28, CTLA-4 or ICOS; (c) increases T-cell proliferationin a Mixed Lymphocyte Reaction (MLR) assay; (d) increases interferon-γproduction in an MLR assay; (e) increases IL-2 secretion in an MLRassay; (f) binds to human PD-1 and cynomolgus monkey PD-1; (g) inhibitsthe binding of PD-L1 and/or PD-L2 to PD-1; (h) stimulatesantigen-specific memory responses; (i) stimulates antibody responses;and/or (j) inhibits tumor cell growth in vivo. Anti-PD-1 antibodiesusable in the present disclosure include monoclonal antibodies that bindspecifically to human PD-1 and exhibit at least one, at least two, atleast three, at least four or at least five of the precedingcharacteristics. In some embodiments, the anti-PD-1 antibody comprisesnivolumab. In one embodiment, the anti-PD-1 antibody comprisespembrolizumab.

In one embodiment, the anti-PD-1 antibody is nivolumab. Nivolumab (alsoknown as “OPDIVO®”; formerly designated 5C4, BMS-936558, MDX-1106, orONO-4538) is a fully human IgG4 (S228P) PD-1 immune checkpoint inhibitorantibody that selectively prevents interaction with PD-1 ligands (PD-L1and PD-L2), thereby blocking the down-regulation of antitumor T-cellfunctions (U.S. Pat. No. 8,008,449; Wang et al., 2014 Cancer ImmunolRes. 2(9):846-56). Nivolumab has shown activity in a variety of advancedsolid tumors including renal cell carcinoma (renal adenocarcinoma, orhypernephroma), melanoma, and non-small cell lung cancer (NSCLC)(Topalian et al., 2012a; Topalian et al., 2014; Drake et al., 2013; WO2013/173223). In another embodiment, the anti-PD-1 antibody (orantigen-binding portion thereof) cross-competes with nivolumab. In someembodiments, the anti-PD-1 antibody binds to the same epitope asnivolumab. In certain embodiments, the anti-PD-1 antibody has the sameCDRs as nivolumab.

In another embodiment, the anti-PD-1 antibody (or antigen-bindingportion thereof) cross-competes with pembrolizumab. In some embodiments,the anti-PD-1 antibody binds to the same epitope as pembrolizumab. Incertain embodiments, the anti-PD-1 antibody has the same CDRs aspembrolizumab. In another embodiment, the anti-PD-1 antibody ispembrolizumab. Pembrolizumab (also known as “KEYTRUDA®”, lambrolizumab,and MK-3475) is a humanized monoclonal IgG4 antibody directed againsthuman cell surface receptor PD-1 (programmed death-1 or programmed celldeath-1). Pembrolizumab is described, for example, in U.S. Pat. No.8,900,587; see also www.cancer.gov/drugdictionary?cdrid=695789 (lastaccessed: Dec. 14, 2014). Pembrolizumab has been approved by the FDA forthe treatment of relapsed or refractory melanoma and advanced NSCLC.

In other embodiments, the anti-PD-1 antibody (or antigen-binding portionthereof) cross-competes with MEDI0680. In some embodiments, theanti-PD-1 antibody binds to the same epitope as MEDI0680. In certainembodiments, the anti-PD-1 antibody has the same CDRs as MEDI0680. Inother embodiments, the anti-PD-1 antibody is MEDI0680 (formerlyAMP-514), which is a monoclonal antibody against the PD-1 receptor.MEDI0680 is described, for example, in U.S. Pat. No. 8,609,089 B2 or inwww.cancer.gov/drugdictionary?cdrid=756047 (last accessed Dec. 14,2014).

In certain embodiments, an immune checkpoint inhibitor is AMP-224, whichis a B7-DC Fc fusion protein. AMP-224 is discussed in U.S. Publ. No.2013/0017199 or inhttp://www.cancer.gov/publications/dictionaries/cancer-drug?cdrid=700595(last accessed Jul. 8, 2015).

In other embodiments, the anti-PD-1 antibody (or antigen-binding portionthereof) cross-competes with BGB-A317. In some embodiments, theanti-PD-1 antibody binds to the same epitope as BGB-A317. In certainembodiments, the anti-PD-1 antibody has the same CDRs as BGB-A317. Incertain embodiments, the anti-PD-1 antibody is BGB-A317, which is ahumanized monoclonal antibody. BGB-A317 is described in U.S. Publ. No.2015/0079109.

In other embodiments, the anti-PD-1 antibody (or antigen-binding portionthereof) cross-competes with INCSHR1210 (SHR-1210). In some embodiments,the anti-PD-1 antibody binds to the same epitope as INCSHR1210(SHR-1210). In certain embodiments, the anti-PD-1 antibody has the sameCDRs as INCSHR1210 (SHR-1210). In certain embodiments, the anti-PD-1antibody is INCSHR1210 (SHR-1210), which is a human monoclonal antibody.INCSHR1210 (SHR-1210) is described in WO2015/085847.

In other embodiments, the anti-PD-1 antibody (or antigen-binding portionthereof) cross-competes with REGN-2810. In some embodiments, theanti-PD-1 antibody binds to the same epitope as REGN-2810. In certainembodiments, the anti-PD-1 antibody has the same CDRs as REGN-2810. Incertain embodiments, the anti-PD-1 antibody is REGN-2810, which is ahuman monoclonal antibody. REGN-2810 is described in WO2015/112800.

In other embodiments, the anti-PD-1 antibody (or antigen-binding portionthereof) cross-competes with PDR001. In some embodiments, the anti-PD-1antibody binds to the same epitope as PDR001. In certain embodiments,the anti-PD-1 antibody has the same CDRs as PDR001. In certainembodiments, the anti-PD-1 antibody is PDR001, which is a humanizedmonoclonal antibody. PDR001 is described in WO2015/112900.

In other embodiments, the anti-PD-1 antibody (or antigen-binding portionthereof) cross-competes with TSR-042 (ANB011). In some embodiments, theanti-PD-1 antibody binds to the same epitope as TSR-042 (ANB011). Incertain embodiments, the anti-PD-1 antibody has the same CDRs as TSR-042(ANB011). In certain embodiments, the anti-PD-1 antibody is TSR-042(ANB011), which is a humanized monoclonal antibody. TSR-042 (ANB011) isdescribed in WO2014/179664.

In other embodiments, the anti-PD-1 antibody (or antigen-binding portionthereof) cross-competes with STI-1110. In some embodiments, theanti-PD-1 antibody binds to the same epitope as STI-1110. In certainembodiments, the anti-PD-1 antibody has the same CDRs as STI-1110. Incertain embodiments, the anti-PD-1 antibody is STI-1110, which is ahuman monoclonal antibody. STI-1110 is described in WO2014/194302.

Anti-PD-1 antibodies usable in the disclosed methods also includeisolated antibodies that bind specifically to human PD-1 andcross-compete for binding to human PD-1 with nivolumab (see, e.g., U.S.Pat. No. 8,008,449; WO 2013/173223). The ability of antibodies tocross-compete for binding to an antigen indicates that these antibodiesbind to the same epitope region of the antigen and sterically hinder thebinding of other cross-competing antibodies to that particular epitoperegion. These cross-competing antibodies are expected to have functionalproperties very similar to those of nivolumab by virtue of their bindingto the same epitope region of PD-1. Cross-competing antibodies can bereadily identified based on their ability to cross-compete withnivolumab in standard PD-1 binding assays such as Biacore analysis,ELISA assays or flow cytometry (see, e.g., WO 2013/173223).

In certain embodiments, the antibodies that cross-compete for binding tohuman PD-1 with, or bind to the same epitope region of human PD-1 asnivolumab are monoclonal antibodies. For administration to humansubjects, these cross-competing antibodies can be chimeric antibodies,or can be humanized or human antibodies. Such chimeric, humanized orhuman monoclonal antibodies can be prepared and isolated by methods wellknown in the art.

Anti-PD-1 antibodies usable in the methods of this disclosure alsoinclude antigen-binding portions of the above antibodies. It has beenamply demonstrated that the antigen-binding function of an antibody canbe performed by fragments of a full-length antibody. Examples of bindingfragments encompassed within the term “antigen-binding portion” of anantibody include (i) a Fab fragment, a monovalent fragment consisting ofthe V_(L), V_(H), C_(L) and C_(H1) domains; (ii) a F(ab′)₂ fragment, abivalent fragment comprising two Fab fragments linked by a disulfidebridge at the hinge region; (iii) a Fd fragment consisting of the V_(H)and C_(H1) domains; (iv) a Fv fragment consisting of the V_(L) and V_(H)domains of a single arm of an antibody; or any combination thereof.

In certain embodiments, the anti-PD-1 antibody or antigen-bindingportion thereof comprises a heavy chain constant region which is of ahuman IgG1 or IgG4 isotype. In certain other embodiments, the sequenceof the IgG4 heavy chain constant region of the anti-PD-1 antibody orantigen-binding portion thereof contains an S228P mutation whichreplaces a serine residue in the hinge region with the proline residuenormally found at the corresponding position in IgG1 isotype antibodies.This mutation, which is present in nivolumab, prevents Fab arm exchangewith endogenous IgG4 antibodies, while retaining the low affinity foractivating Fc receptors associated with wild-type IgG4 antibodies (Wanget al., 2014). In yet other embodiments, the antibody comprises a lightchain constant region which is a human kappa or lambda constant region.In other embodiments, the anti-PD-1 antibody or antigen-binding portionthereof is a monoclonal antibody or an antigen-binding portion thereof.In certain embodiments of any of the therapeutic methods describedherein comprising administration of an anti-PD-1 antibody, the anti-PD-1antibody is nivolumab. In other embodiments, the anti-PD-1 antibody ispembrolizumab. In other embodiments, the anti-PD-1 antibody is chosenfrom the human antibodies 17D8, 2D3, 4H1, 4A11, 7D3 and 5F4 described inU.S. Pat. No. 8,008,449. In still other embodiments, the anti-PD-1antibody is MEDI0680 (formerly AMP-514), AMP-224, or BGB-A317.

In other embodiments, the anti-PD-1 antibody or antigen-binding portionthereof is a chimeric, humanized or human monoclonal antibody or aportion thereof. In certain embodiments for treating a human subject,the antibody is a humanized antibody. In other embodiments for treatinga human subject, the antibody is a human antibody. Antibodies of anIgG1, IgG2, IgG3 or IgG4 isotype may be used.

In certain embodiments, an anti-PD-1 antibody used in the methods can bereplaced with another PD-1 or anti-PD-L1 antagonist. For example,because an anti-PD-L1 antibody prevents interaction between PD-1 andPD-L1, thereby exerting similar effects to the signaling pathway ofPD-1, an anti-PD-L1 antibody can replace the use of an anti-PD-1antibody in the methods disclosed herein. Therefore, in one embodiment,the present disclosure is directed to a method for treating a subjectafflicted with a tumor derived from a colorectal cancer comprisingadministering to the subject a therapeutically effective amount of ananti-cancer agent which is an antibody or an antigen-binding portionthereof that binds specifically to a Programmed Death-Ligand 1 (PD-L1)receptor and inhibits PD-L1 activity (an anti-PD-L1 antibody). In otherembodiments, the present disclosure is directed to a method for treatinga subject afflicted with a tumor derived from a colorectal cancercomprising administering to the subject a therapeutically effectiveamount of an anti-PD-L1 antibody in combination with an anti-CTLA-4antibody. In certain embodiments, the anti-PD-L1 Ab is BMS-936559(formerly 12A4 or MDX-1105) (see, e.g., U.S. Pat. No. 7,943,743; WO2013/173223). In other embodiments, the anti-PD-L1 Ab is MPDL3280A (alsoknown as RG7446 or atezolizumab) (see, e.g., Herbst; U.S. Pat. No.8,217,149), MEDI4736 (also called Durvalumab; Khleif, 2013, See U.S.Pat. No. 8,779,108 or US 2014/0356353, filed May 6, 2014), orMSB0010718C (also called Avelumab; See US 2014/0341917). In otherembodiments, the anti-PD-L1 antibody is CX-072 (also called CytomX; SeeWO2016/149201)

Anti-CTLA-4 Antibodies

In some embodiments, the methods of the present disclosure can be acombination therapy of an anti-PD-1 antibody or an anti-PD-L1 antibodyand an anti-CTLA-4 antibody. Anti-CTLA-4 antibodies of the instantdisclosure bind to human CTLA-4 so as to disrupt the interaction ofCTLA-4 with a human B7 receptor. Because the interaction of CTLA-4 withB7 transduces a signal leading to inactivation of T-cells bearing theCTLA-4 receptor, disruption of the interaction effectively induces,enhances or prolongs the activation of such T cells, thereby inducing,enhancing or prolonging an immune response.

Human monoclonal antibodies that bind specifically to CTLA-4 with highaffinity have been disclosed in U.S. Pat. Nos. 6,984,720 and 7,605,238.Other anti-PD-1 monoclonal antibodies have been described in, forexample, U.S. Pat. Nos. 5,977,318, 6,051,227, 6,682,736, and 7,034,121.The anti-PD-1 human monoclonal antibodies disclosed in U.S. Pat. Nos.6,984,720 and 7,605,238 have been demonstrated to exhibit one or more ofthe following characteristics: (a) binds specifically to human CTLA-4with a binding affinity reflected by an equilibrium association constant(K_(a)) of at least about 10⁷ M⁻¹, or about 10⁹ M⁻¹, or about 10¹⁰ M⁻¹to 10¹¹ M⁻¹ or higher, as determined by Biacore analysis; (b) a kineticassociation constant (k_(a)) of at least about 10³, about 10⁴, or about10⁵ m⁻¹ s⁻¹; (c) a kinetic disassociation constant (k_(d)) of at leastabout 10³, about 10⁴, or about 10⁵ m⁻¹ s⁻¹; and (d) inhibits the bindingof CTLA-4 to B7-1 (CD80) and B7-2 (CD86). Anti-CTLA-4 antibodies usefulfor the present disclosure include monoclonal antibodies that bindspecifically to human CTLA-4 and exhibit at least one, at least two, orat least three of the preceding characteristics.

An exemplary clinical anti-CTLA-4 antibody is the human monoclonalantibody 10D1 (now known as ipilimumab and marketed as YERVOY®) asdisclosed in U.S. Pat. No. 6,984,720. Ipilimumab is an anti-CTLA-4antibody for use in the methods disclosed herein. Ipilimumab is a fullyhuman, IgG1 monoclonal antibody that blocks the binding of CTLA-4 to itsB7 ligands, thereby stimulating T cell activation and improving overallsurvival (OS) in patients with advanced melanoma.

Another anti-CTLA-4 antibody useful for the present methods istremelimumab (also known as CP-675,206). Tremelimumab is human IgG2monoclonal anti-CTLA-4 antibody. Tremelimumab is described inWO/2012/122444, U.S. Publ. No. 2012/263677, or WO Publ. No. 2007/113648A2.

Anti-CTLA-4 antibodies useful for the disclosed composition also includeisolated antibodies that bind specifically to human CTLA-4 andcross-compete for binding to human CTLA-4 with ipilimumab ortremelimumab or bind to the same epitope region of human CTLA-4 asipilimumab or tremelimumab. In certain embodiments, the antibodies thatcross-compete for binding to human CTLA-4 with, or bind to the sameepitope region of human CTLA-4 as does ipilimumab or tremelimumab, areantibodies comprising a heavy chain of the human IgG1 isotype. Foradministration to human subjects, these cross-competing antibodies arechimeric antibodies, or humanized or human antibodies. Usefulanti-CTLA-4 antibodies also include antigen-binding portions of theabove antibodies such as Fab, F(ab′)₂, Fd, or Fv fragments.

Cancer and Standard-of-Care Therapies

In some embodiments, the methods disclosed herein are used in place ofstandard of care therapies. In certain embodiments, a standard of caretherapy is used in combination with any method disclosed herein.Standard-of-care therapies for different types of cancer are well knownby persons of skill in the art. For example, the National ComprehensiveCancer Network (NCCN), an alliance of 21 major cancer centers in theUSA, publishes the NCCN Clinical Practice Guidelines in Oncology (NCCNGUIDELINES®) that provide detailed up-to-date information on thestandard-of-care treatments for a wide variety of cancers (see NCCNGUIDELINES®, 2014).

Pharmaceutical Compositions and Dosages

Therapeutic agents of the present disclosure can be constituted in acomposition, e.g., a pharmaceutical composition containing an antibodyand a pharmaceutically acceptable carrier. As used herein, a“pharmaceutically acceptable carrier” includes any and all solvents,dispersion media, coatings, antibacterial and antifungal agents,isotonic and absorption delaying agents, and the like that arephysiologically compatible. In some embodiments, the carrier for acomposition containing an antibody is suitable for intravenous,intramuscular, subcutaneous, parenteral, spinal or epidermaladministration (e.g., by injection or infusion). A pharmaceuticalcomposition of the disclosure can include one or more pharmaceuticallyacceptable salts, anti-oxidant, aqueous and non-aqueous carriers, and/oradjuvants such as preservatives, wetting agents, emulsifying agents anddispersing agents.

Dosage regimens are adjusted to provide the optimum desired response,e.g., a maximal therapeutic response and/or minimal adverse effects. Insome embodiments, the anti-PD-1 antibody is administered at aweight-based dose. For administration of an anti-PD-1 antibody, thedosage can range from at least about 0.01 to at least about 20 mg/kg,from at least about 0.1 to at least about 10 mg/kg, of the subject'sbody weight. For example, dosages can be at least about 0.1 mg/kg, atleast about 0.3 mg/kg, at least about 1 mg/kg, at least about 2 mg/kg,at least about 3 mg/kg, at least about 5 mg/kg, or at least about 10mg/kg body weight. In certain embodiments, the dosage of the anti-PD-1antibody is 3 mg/kg body weight. In certain embodiments, an anti-PD-1antibody is administered at a flat dose. In certain embodiments, theflat dose of the anti-PD-1 antibody is a dose (e.g., flat dose) of atleast about 100-300 mg, such as, at least about 200-300 mg, at leastabout 220-260 mg, at least about 230-250 mg or at least about 240 mg,such as at least about 60 mg, at least about 80 mg, at least about 100mg, at least about 120 mg, at least about 140 mg, at least about 160 mg,at least about 180 mg, at least about 200 mg, at least about 220 mg, atleast about 240 mg, at least about 260 mg, at least about 280 mg or atleast about 300 mg. In some embodiments, the anti-PD-1 antibody isadministered in a fixed dose with the anti-CTLA-4 antibody. In someembodiments, the ratio is at least about 1:1, about 1:2, about 1:3,about 1:4, about 1:5, about 1:6, about 1:7, about 1:8, about 1:9, about1:10, about 1:15, about 1:20, about 1:30, about 1:40, about 1:50, about1:60, about 1:70, about 1:80, about 1:90, about 1:100, about 1:120,about 1:140, about 1:160, about 1:180, about 1:200, about 200:1, about180:1, about 160:1, about 140:1, about 120:1, about 100:1, about 90:1,about 80:1, about 70:1, about 60:1, about 50:1, about 40:1, about 30:1,about 20:1, about 15:1, about 10:1, about 9:1, about 8:1, about 7:1,about 6:1, about 5:1, about 4:1, about 3:1, or about 2:1 mg anti-PD-1antibody to mg anti-CTLA-4 antibody.

The dosing schedule is typically designed to achieve exposures thatresult in sustained receptor occupancy (RO) based on typicalpharmacokinetic properties of an antibody. An exemplary treatment regimeentails administration once per week, once about every 2 weeks, onceabout every 3 weeks, once about every 4 weeks, once about a month, onceabout every 3-6 months or longer. In certain embodiments, an anti-PD-1antibody such as nivolumab is administered to the subject once aboutevery 2 weeks. In other embodiments, the antibody is administered onceabout every 3 weeks. The dosage and scheduling can change during acourse of treatment.

When used in combinations with other anti-cancer agents, the dosage ofan anti-PD-1 antibody can be lowered compared to the monotherapy dose.Dosages of nivolumab that are lower than the typical 3 mg/kg, but notless than 0.001 mg/kg, are subtherapeutic dosages. The subtherapeuticdoses of an anti-PD-1 antibody used in the methods herein are higherthan 0.001 mg/kg and lower than 3mg/kg. In some embodiments, asubtherapeutic dose is about 0.001 mg/kg to about 1 mg/kg, about 0.01mg/kg to about 1 mg/kg, about 0.1 mg/kg to about 1 mg/kg, or about 0.001mg/kg to about 0.1 mg/kg body weight. In some embodiments, thesubtherapeutic dose is at least about 0.001 mg/kg, at least about 0.005mg/kg, at least about 0.01 mg/kg, at least about 0.05 mg/kg, at leastabout 0.1 mg/kg, at least about 0.5 mg/kg, or at least about 1.0 mg/kgbody weight. Receptor-occupancy data from 15 subjects who received 0.3mg/kg to 10 mg/kg dosing with nivolumab indicate that PD-1 occupancyappears to be dose-independent in this dose range. Across all doses, themean occupancy rate was 85% (range, 70% to 97%), with a mean plateauoccupancy of 72% (range, 59% to 81%). In some embodiments, 0.3 mg/kgdosing can allow for sufficient exposure to lead to maximal biologicactivity.

In some embodiments, the anti-CTLA-4 antibody is administered at aweight-based dose. For administration of an anti-CTLA-4 antibody thedosage can range from about 0.01 to about 20 mg/kg, about 0.05 to about20 mg/kg, about 0.1 to about 20 mg/kg, about 0.1 to about 15 mg/kg,about 0.1 to about 10 mg/kg, about 0.1 to about 5 mg/kg, and about 1 toabout 10 mg/kg of the subject's body weight. For example, dosages can beabout 0.05 mg/kg, about 0.1 mg/kg, about 0.5 mg/kg, about 1 mg/kg, about2 mg/kg, about 3 mg/kg, about 4 mg/kg, about 5 mg/kg, about 6 mg/kg,about 7 mg/kg, about 8 mg/kg, about 9 mg/kg, about 10 mg/kg, about 11mg/kg, about 12 mg/kg, about 13 mg/kg, about 14 mg/kg, about 15 mg/kg orabout 20 mg/kg of the subject's body weight. In some embodiments, thedosage of the anti-CTLA-4 antibody is 0.1 mg/kg body weight. In otherembodiments, the dosage of the anti-CTLA-4 antibody is 1 mg/kg bodyweight. In further embodiments, the dosage of the anti-CTLA-4 antibodyis 10 mg/kg body weight. In certain embodiments, an anti-CTLA-4 antibodyis administered at a flat dose. In embodiments, the flat dose of theanti-CTLA-4 is a dose (e.g., flat dose) of at least about 60-1500 mg,such as, at least about 100-1400 mg, at least about 100-1000 mg, atleast about 200-1000 mg or at least about 200-500 mg, such as at leastabout 60 mg, at least about 80 mg, at least about 100 mg, at least about120 mg, at least about 140 mg, at least about 160 mg, at least about 180mg, at least about 200 mg, at least about 220 mg, at least about 240 mg,at least about 260 mg, at least about 280 mg, at least about 300 mg, atleast about 320 mg, at least about 340 mg, at least about 360 mg, atleast about 380 mg, at least about 400 mg, at least about 420 mg, atleast about 440 mg, at least about 460 mg, at least about 480 mg, atleast about 500 mg, at least about 600 mg, at least about 700 mg, atleast about 800 mg, at least about 900 mg, at least about 1000 mg, atleast about 1100 mg, at least about 1200 mg, at least about 1300 mg, atleast about 1400 mg, or at least about 1500 mg.

An exemplary treatment regime entails administration once per week, onceabout every 2 weeks, once about every 3 weeks, once about every 4 weeks,once about a month, once about every 3-6 months or longer. In certainembodiments, the anti-CTLA-4 antibody is administered once about every 3weeks.

In some embodiments, a subtherapeutic dose of an anti-CTLA-4 antibody isused in the methods herein. The subtherapeutic dosages of an anti-CTLA-4antibody used in the methods herein are higher than 0.001 mg/kg andlower than 10 mg/kg. In some embodiments, the subtherapeutic dose isabout 0.001 mg/kg to about 10 mg/kg, about 0.01 mg/kg to about 10 mg/kg,about 0.01 mg/kg to about 1 mg/kg, about 0.1 mg/kg to about 1 mg/kg, orabout 0.001 mg/kg to about 0.1 mg/kg body weight. In some embodiments,the subtherapeutic dose is at least about 0.001 mg/kg, at least about0.005 mg/kg, at least about 0.01 mg/kg, at least about 0.05 mg/kg, atleast about 0.1 mg/kg, at least about 0.5 mg/kg, at least about 1.0mg/kg, at least about 2 mg/kg, at least about 3 mg/kg, at least about 4mg/kg, at least about 5 mg/kg, at least about 6 mg/kg, at least about 7mg/kg, at least about 8 mg/kg, at least about 9 mg/kg, or at least about10 mg/kg body weight. In some embodiments, the subtherapeutic dose isabout 10 mg/kg, about 5 mg/kg, about 2 mg/kg, about 1 mg/kg, about 0.1mg/kg, or about 0.01 mg/kg body weight.

In certain embodiments, at least about 0.1 to about 10 mg/kg of theanti-CTLA-4 antibody and at least about 0.1 to about 10 mg/kg of theanti-PD1 antibody are administered to the subject once about every threeweeks. In certain embodiments, at least about 1 mg/kg of the anti-CTLA-4antibody and at least about 1 mg/kg of the anti-PD1 antibody areadministered to the subject once about every three weeks. In certainembodiments, at least about 1 mg/kg of the anti-CTLA-4 antibody and atleast about 3 mg/kg of the anti-PD1 antibody are administered to thesubject once about every three weeks. In certain embodiments, at leastabout 3 mg/kg of the anti-CTLA-4 antibody and at least about 1 mg/kg ofthe anti-PD-1 antibody are administered to the subject one about everythree weeks. In certain embodiments, at least about 3 mg/kg of theanti-CTLA-4 antibody and at least about 3 mg/kg of the anti-PD1 antibodyare administered to the subject once about every three weeks. Inembodiments, the anti-CTLA-4 antibody is ipilimumab. In someembodiments, the anti-PD-1 antibody is nivolumab.

In certain embodiments, the combination of an anti-PD-1 antibody and ananti-CTLA-4 antibody is administered intravenously to the subject onceabout every 3 weeks for a total of 12 weeks. In some embodiments, the 12week cycle is repeated 3 or 4 times. In some embodiments, the subject istreated with a combination of an anti-PD-1 antibody and an anti-CTLA-4antibody every 3 weeks for a total of 12 weeks and 3 twelve-week cyclesare performed. In embodiments, the subject is treated with a combinationof an anti-PD-1 antibody and an anti-CTLA-4 antibody every 3 weeks for atotal of 12 weeks and 4 twelve-week cycles are performed. Inembodiments, a subject is treated with the anti-PD1 antibody for 12twelve-week cycles.

In certain embodiments, the administration of an anti-PD-1 antibody andan anti-CTLA-4 antibody is followed by an anti-PD-1 antibodymonotherapy. In some embodiments, the antibody monotherapy after thecombination therapy of an anti-PD-1 antibody and an anti-CTLA-4 antibodycomprises administering an anti-PD-1 antibody at a dose of about 1 mg/kgbody weight, about 2 mg/kg body weight, about 3 mg/kg body weight, about4 mg/kg body weight, about 5 mg/kg body weight, about 6 mg/kg bodyweight, about 7 mg/kg body weight, about 8 mg/kg body weight, about 9mg/kg body weight, or about 10 mg/kg body weight. In some embodiments,the anti-PD-1 monotherapy provided after the combination therapy of ananti-PD-1 antibody and an anti-CTLA-4 antibody is administered onceabout every 1, 2, 3, or 4 weeks. In a certain embodiment, the anti-PD-1antibody monotherapy provided after the combination therapy of ananti-PD-1 antibody and an anti-CTLA-4 antibody is administered at a doseof about 3 mg/kg body weight once every 2 weeks.

In other embodiments, the anti-PD-1 antibody is administered at 3 mg/kgonce every 3 weeks and the anti-CTLA-4 antibody is administered at 1mg/kg once every 3 weeks. In certain embodiments, the anti-PD-1 antibodyand the anti-CTLA-4 antibody are administered in combination for onedose each, two doses for each, three doses for each, four doses foreach, five doses for each, six doses for each, seven doses for each,eight doses for each, nine doses for each, or ten doses for each. In aparticular embodiment, the anti-PD-1 antibody and the anti-CTLA-4antibody are administered in combination for four doses for each. Incertain embodiments, the subject is administered, prior to and/or afterthe combination therapy of the anti-PD-1 antibody and the anti-CTLA-4antibody, an anti-PD-1 antibody at a dose of 3 mg/kg for once every 2weeks. In a particular embodiment, the subject afflicted with acolorectal cancer with MSI-H is treated with an initial stage (e.g., ananti-PD-1 antibody monotherapy, e.g., nivolumab at a dose of 3 mg/kgonce every two weeks), a combination stage (e.g., an anti-PD-1 antibodyand an anti-CTLA-4 antibody combination, e.g., nivolumab at a dose of 3mg/kg once every three weeks and ipilimumab at a dose of 1 mg/kg onceevery week), and a final stage (e.g., an anti-PD-1 antibody monotherapy,e.g., nivolumab at a dose of 3 mg/kg once every two weeks).

Treatment can be continued as long as clinical benefit is observed oruntil unacceptable toxicity or disease progression occurs. In certainembodiments, the anti-PD-1 antibody can be administered at the dosagethat has been shown to produce the highest efficacy as monotherapy inclinical trials, e.g., about 3 mg/kg of nivolumab administered onceabout every three weeks (Topalian et al., 2012 N Engl J Med 366:2443-54;Topalian et al., 2012 Curr Opin Immunol 24:207-12), or at asignificantly lower dose, i.e., at a subtherapeutic dose.

Actual dosage levels of the active ingredients in the pharmaceuticalcompositions of the present disclosure can be varied so as to obtain anamount of the active ingredient which is effective to achieve thedesired therapeutic response for a particular patient, composition, andmode of administration, without being unduly toxic to the patient. Theselected dosage level will depend upon a variety of pharmacokineticfactors including the activity of the particular compositions of thepresent disclosure employed, the route of administration, the time ofadministration, the rate of excretion of the particular compound beingemployed, the duration of the treatment, other drugs, compounds and/ormaterials used in combination with the particular compositions employed,the age, sex, weight, condition, general health and prior medicalhistory of the patient being treated, and like factors well known in themedical arts. A composition of the present disclosure can beadministered via one or more routes of administration using one or moreof a variety of methods well known in the art. As will be appreciated bythe skilled artisan, the route and/or mode of administration will varydepending upon the desired results.

Kits

Also within the scope of the present disclosure are kits comprising ananti-PD-1 antibody and another anti-cancer agent for therapeutic uses.Kits typically include a label indicating the intended use of thecontents of the kit and instructions for use. The term label includesany writing, or recorded material supplied on or with the kit, or whichotherwise accompanies the kit. Accordingly, this disclosure provides akit for treating a subject afflicted with a tumor derived from acolorectal cancer, the kit comprising: (a) a dosage ranging from about0.1 mg/kg to about 10 mg/kg of an anti-PD-1 antibody or antigen-bindingportion thereof; and (b) instructions for using the anti-PD-1 antibodyin any of the monotherapy methods disclosed herein. In otherembodiments, the kit for treating a subject afflicted with a tumorderived from a colorectal cancer comprises: (a) a dosage ranging fromabout 0.1 mg/kg to about 10 mg/kg of an anti-PD-1 antibody orantigen-binding portion thereof; and (b) a dosage ranging from about 0.1mg/kg to about 10 mg/kg of an anti-CTLA-4 antibody or antigen-bindingportion thereof and (c) instructions for using the anti-PD-1 antibodyand the anti-CTLA-4 antibody in any of the combination therapy methodsdisclosed herein. In certain embodiments, the anti-PD-1 antibody and theanti-CTLA-4 antibody can be co-packaged in unit dosage form. In certainembodiments for treating human patients, the kit comprises an anti-humanPD-1 antibody disclosed herein, e.g., nivolumab, pembrolizumab, MEDI0608(formerly AMP-514), AMP-224, or BGB-A317. In other embodiments, the kitcomprises an anti-human CTLA-4 antibody disclosed herein, e.g.,ipilimumab or tremelimumab.

The present disclosure is further illustrated by the following exampleswhich should not be construed as further limiting. The contents of allreferences cited throughout this application are expressly incorporatedherein by reference.

Cross-reference to earlier filed applications: the present applicationclaims benefit to U.S. provisional application No. 62/345,662 filed Jun.3, 2016, which is incorporated by reference herein by reference in itsentirety.

EXAMPLES Example 1

Clinical Case Study of Nivolumab+Ipilimumab in the Treatment of Patientswith Metastatic Colorectal Cancer with and without High MicrosatelliteInstability

Patients were eligible for inclusion in this study based upon thefollowing criteria:

1) histologically confirmed colorectal cancer; 2) recurrent ormetastatic disease measurable by Response Evaluation Criteria in SolidTumors (RECIST) 1.1; 3) patient is an adult at least 18 years of agewith Eastern Cooperative Oncology Group (ECOG) scores of 0-1; and 4)disease progression after ≥1 prior therapy regimen (for microsatelliteinstability-high patients) or after the latest treatment (for allpatients) or intolerance or refusal to take chemotherapy. Patients wereexcluded from the study based upon the following criteria: 1) centralnervous system involvement; 2) history of malignancy within 3 years; 3)active or history of autoimmune disease; 4) need for treatment withimmunosuppressive medications including corticosteroids; or 5) priortreatments targeting T-cell costimulation or immune checkpoint pathways.

The colorectal cancer of patients was determined to be microsatelliteinstability-high (MSI-H) by polymerase chain reaction (PCR) if two ormore markers showed instability out of 5 loci tested or if at least 30%of the markers showed instability when 5 or more loci were tested. Thecolorectal cancer of patients was also determined to be MSI-H byimmunohistochemistry (IHC) if there was a loss of one or more markers.

Patients with 3L or later colon cancer that is non-MSI-H and with anECOG performance status of 0-1 were selected for inclusion in themicrosatellite stable (MSS) cohort of patients. Selected MSS patientswere administered 1 mg/kg nivolumab in combination with 1 mg/kgipilimumab once every 3 weeks for a total of 4 doses, followed by 3mg/kg nivolumab once every 2 weeks. Six or more weeks following thisinitial phase of treatment, MSS patients who tolerated the initialtherapeutic regimen were split into two treatment groups. In onetreatment group, 10 MSS patients were administered 1 mg/kg nivolumab incombination with 3 mg/kg ipilimumab once every 3 weeks for a total of 4doses, followed by 3 mg/kg nivolumab once every 2 weeks. In a secondtreatment group, 10 MSS patients were administered 3 mg/kg nivolumab incombination with 1 mg/kg ipilimumab once every 3 weeks for a total of 4doses, followed by 3 mg/kg once every 2 weeks. Collectively, thesetreatment groups served as the independent safety arm in MSS patientsand informed the dose of nivolumab in combination with ipilimumab foruse in MSI-H patients. FIG. 1 shows the overall study design forpatients in the MSS cohort.

Patients with 2^(nd) MSI-H colon cancer who had received at least 1prior treatment for metastatic disease, who had at least 1 targetlesion, and who had an ECOG performance status of 0-1 were selected forinclusion in the MSI-H cohort of patients. Nineteen MSI-H patients wereadministered 3 mg/kg nivolumab once every 2 weeks in an initialmonotherapy stage of treatment (mStage 1). Certain MSI-H patientsexhibiting positive responses were then selected for inclusion in acombination therapy stage of treatment (cStage 1). Nineteen of theseMSI-H patients were then administered 3 mg/kg nivolumab in combinationwith 1 mg/kg ipilimumab once every 3 weeks for a total of 4 doses,followed by 3 mg/kg nivolumab once every two weeks in cStage 1. A numberof patients with positive responses from the cStage 1 are currentlybeing enrolled in a second stage of combination therapy (cStage 2). IncStage 2, 48 patients (19 patients from cStage 1 plus 29 additionalpatients) will receive 3 mg/kg nivolumab in combination with 1 mg/kgipilimumab once every 3 weeks for a total of 4 doses, followed by 3mg/kg nivolumab once every two weeks. A separate group of patientsexhibiting positive responses in mStage 1 were selected for inclusion ina second stage of monotherapy (mStage 2). In mStage 2, 48 patients (19patients from mStage 1 plus 29 new patients) were administered 3 mg/kgnivolumab once every 2 weeks. FIG. 2 shows the overall study design forpatients in the MSI-H cohort.

The primary endpoint of this study was to determine theinvestigator-assessed objective response rate (ORR) using RECIST 1.1 inMSI-H patients. The secondary endpoint of this study was to determinethe independent radiology review committee-assessed ORR in patients.Exploratory endpoints of this study included an assessment of safety andtolerability, progression-free survival (PFS), overall survival (OS),investigator-assessed ORR in MSS patients, and biomarkers.

The patient demographics collected for this study included median age,the number of patients under age 65, the number of male patients, thenumber of patients of a particular race, and the number of patients withan ECOG score of 0 or 1. Table 1 shows the demographics for MSI-Hpatients receiving 3 mg/kg nivolumab monotherapy compared to MSI-Hpatients receiving 3 mg/kg nivolumab in combination with 1 mg/kgipilimumab. Table 2 also shows the demographics for MSS patientsreceiving 1 mg/kg nivolumab in combination with 3 mg/kg ipilimumabcompared to MSS patients receiving 3 mg/kg nivolumab in combination with1 mg/kg ipilimumab.

TABLE 2 Demographics for patients with MSI-H or MSS colorectal cancer.MSI-H MSS Nivo 3 Nivo l Nivo 3 Nivo3 mg/kg + mg/kg + mg/kg + mg/kg Ipi 1mg/kg Ipi 3 mg/kg Ipi 1 mg/kg (n = 70)^(a) (n = 30)^(b) (n = 10) (n =10) Median age, 53 (26-79) 60 (33-81) 49 (35-65) 52 (38-69) years(range) Age, <65 years, 54 (77.1) 22 (73.3)  9 (90.0)  9 (90.0) n (%)    Male, n (%) 42 (60.0) 15 (50.0)  7 (70.0)  8 (80.0) Race, n (%)    White 61 (87.1) 25 (83.3)  10(100)  10(100) Black  7 (10.0)  1 (3.3) 0 0Asian  1 (1.4)  2 (6.7) 0 0 Other  1 ( 1.4)  2 (6.7) 0 0 ECOG score, n(%)c 0 30 (42.9) 10 (33.3)  4 (40.0)  3 (30.0) 1 39 (55.7) 20 (66.7)  6(60.0)  7 (70.0) ECOG, Eastern Cooperative Oncology Group. ^(a)mStages 1and 2 combined; ^(b)cStages 1 and 2 combined; ^(c)One patient with anECOG score of 1 at randomization had deteriorated to a score of 3 by thetime of treatment initiation

Data collected on disease characteristics and prior therapy included thenumber of patients with a particular disease stage at diagnosis, thenumber of patients with a particular mutation status, the number ofpatients receiving prior treatments, the number of patients receivingprior surgery, and the number of patients receiving prior radiotherapy.Table 3 shows the disease characteristics and prior therapy for MSI-Hpatients receiving 3 mg/kg nivolumab monotherapy compared to MSI-Hpatients receiving 3 mg/kg nivolumab in combination with 1 mg/kgipilimumab. Table 3 also shows the disease characteristics and priortherapy for MSS patients receiving 1 mg/kg nivolumab in combination with3 mg/kg ipilimumab compared to MSS patients receiving 3 mg/kg nivolumabin combination with 1 mg/kg ipilimumab.

TABLE 3 Disease characteristics and prior therapy for patients withMSI-H or MSS colorectal cancer. MSI-H MSS Nivolumab 3 Nivolumab 1Nivolumab 3 mg/kg + mg/kg + mg/kg + Nivolumab Ipilimumab IpilimumabIpilimumab 3 mg/kg 1 mg/kg 3 mg/kg 1 mg/kg (n = 70)^(a) (n = 30)^(b) (n= 10) (n = 10) Disease stage at diagnosis, n (%) I-II 15 (21.4)  2 (6.7)1 (10.0) 1 (10.0) III-IV 54 (77.1) 28 (93.3) 9 (90.0) 9 (90.0) Mutationstatus, n (%) KRAS/BRAF wild 26 (37.1)  6 (20.0) 2 (20.0) 5 (50.0) typeBRAF mutated 11 (15.7)  6 (20.0) 0 0 KRAS mutated 23 (32.9) 14 (46.7) 6(60.0) 3 (30.0) Unknown 10 (14.3)  4 (13.3) 2 (20.0) 2 (20.0) Priortreatments, n (%) 1  9 (12.9)  2 (6.7) — — 2 21 (30.0) 15 (50.0) ≥3 39(55.7) 13 (43.3) Prior surgery, n 70 (100) 30 (100) — — (%) Priorradiotherapy, 26 (37.1) 7 (23.3) — — n (%) ^(a)mStages 1 and 2 combined;^(b)cStages 1 and 2 combined

Data regarding the dispositions of MSI-H patients collected in thisstudy included the number of patients continuing treatment, the numberof patients not continuing treatment, and the number of patients notcontinuing treatment due to disease progression, study drug toxicity,withdrawal of consent or other reasons, or for unreported reasons. Table4 shows the patient dispositions for MSI-H patients receiving 3 mg/kgnivolumab monotherapy compared to MSI-H patients receiving 3 mg/kgnivolumab in combination with 1 mg/kg ipilimumab.

TABLE 4 MSI-H patient disposition. MSI-H Nivolumab Nivolumab 3 mg/kg + 3mg/kg Ipilimumab 1 mg/kg (n = 70) (n = 30) Continuing treatment, n (%)47 (67.1) 18 (60.0) Not continuing treatment, n (%) 23 (32.9) 12 (40.0)Reasons for not continuing, n (%) Disease progression 19 (27.1)  6(20.0) Study drug toxicity  2 (2.9)  4 (13.3) Withdrew consent/Other  1(1.4)  1 (3.3) Not reported  1 (1.4)  1 (3-3)

The investigator-assessed best overall response in MSI-H patientsreceiving 3 mg/kg nivolumab monotherapy was determined by the objectiveresponse rate, the median time to response, and the median duration ofresponse. As shown in Table 5, 12 out of 47 patients (25.5%) exhibitedobjective responses to 3 mg/kg nivolumab monotherapy, with the mediantime to response occurring at 2.12 months following treatment. FIG. 3shows the percent change from baseline for individual patients receiving3 mg/kg nivolumab monotherapy.

TABLE 5 Investigator-assessed best overall response in MSI-H patientsreceiving nivolumab monotherapy. Nivolumab 3 mg/kg (n = 47)^(a)Objective response rate, n (%) 12/47 (25.5) (95% exact CI) (15.4, 38.1)Complete response 0 Partial response 12 (25.5) Stable disease 14 (29.8)Progressive disease 17 (36.2) Unable to determine 4 (8.5) Median time toresponse, mo (range) 2.12 (1.3-13.6) Median duration of response, mo(range) NE (0.0^(b)-15.2^(b)) ^(a)Patients with ≥12 weeks of follow-up^(b)Includes censored observations

The investigator-assessed best overall response in MSI-H patientsreceiving 3 mg/kg nivolumab in combination with 1 mg/kg ipilimumab wasdetermined by the objective response rate, the median time to response,and the median duration of response. As shown in Table 6, 9 out of 27patients (33.3%) exhibited objective responses to treatment with 3 mg/kgnivolumab in combination with 1 mg/kg ipilimumab, with the median timeto response occurring at 2.73 months following treatment. FIG. 4 showsthe percent change from baseline for individual patients receiving 3mg/kg nivolumab in combination with 1 mg/kg ipilimumab.

TABLE 6 Investigator-assessed best overall response in MSI-H patientsreceiving nivolumab in combination with ipilimumab. Nivolumab 3 mg/kg +Ipilimumab 1 mg/kg (n = 27)^(a) Objective response rate, n (%) 9/27(33.3) (95% exact CI) (18.6, 50.9) Complete response 0 Partial response9 (33.3) Stable disease 14(51.9) Progressive disease 3 (11.1) Unable todetermine 0 Median time to response, mo 2.73 (1.2-6.9) (range) Medianduration of response, mo NE (NE-NE) (range) ^(a)Patients with ≥12 weeksof follow-up ^(b)Includes censored observations

The best reduction in target lesion size for MSI-H patients receiving 3mg/kg nivolumab monotherapy was compared to the best reduction in targetlesion size for MSI-H patients receiving 3 mg/kg nivolumab incombination with 1 mg/kg ipilimumab. As shown in FIG. 5, 56% of patientsreceiving 3 mg/kg nivolumab monotherapy exhibited a reduction in targetlesion size from baseline, while 81% of patients receiving 3 mg/kgnivolumab in combination with 1 mg/kg ipilimumab exhibited a reductionin target lesion size from baseline.

Investigator-assessed PFS was measured at 6 months, 9 months, and 12months for MSI-H patients receiving 3 mg/kg nivolumab monotherapy and at6 months for MSI-H patients receiving 3 mg/kg nivolumab in combinationwith 1 mg/kg ipilimumab. As shown in Table 7, 45.9% of patientsreceiving 3 mg/kg nivolumab monotherapy exhibited progression-freesurvival at 6 months, while 66.6% of patients receiving 3 mg/kgnivolumab in combination with 1 mg/kg ipilimumab exhibitedprogression-free survival at 6 months. FIG. 6 shows the percentage ofpatients exhibiting progression-free survival for those receiving 3mg/kg nivolumab monotherapy compared to those receiving 3 mg/kgnivolumab in combination with 1 mg/kg ipilimumab.

TABLE 7 Investigator-assessed progression-free survival in MSI-Hpatients. Nivo 3 mg/kg + Nivolumab Ipilimumab 3 mg/kg 1 mg/kg (n = 70)(n = 30) PFS rate, % (95% CI) 6 mo 45.9 (29.8, 60.7) 66.6 (45.5, 81.1)mo 45.9 (29.8, 60.7) NE 12 mo 45.9 (29.8, 60.7) NE Median PFS, mo (95%CI)  5.3 (1.5, NE) NE (3.4, NE)

OS was measured at 6 months, 9 months, and 12 months for MSI-H patientsreceiving 3 mg/kg nivolumab monotherapy and at 6 months and 9 months forMSI-H patients receiving 3 mg/kg nivolumab in combination with 1 mg/kgipilimumab. As shown in Table 8, 75.0% of patients receiving 3 mg/kgnivolumab monotherapy exhibited progression-free survival at 6 months,while 85.1% of patients receiving 3 mg/kg nivolumab in combination with1 mg/kg ipilimumab exhibited progression-free survival at 6 months. At 9months, 65.6% of patients receiving 3 mg/kg nivolumab monotherapyexhibited progression-free survival, while 85.1% of patients receiving 3mg/kg nivolumab in combination with 1 mg/kg ipilimumab exhibitedprogression free survival. FIG. 7 shows the percentage of patientsexhibiting overall survival for those receiving 3 mg/kg nivolumabmonotherapy compared to those receiving 3 mg/kg nivolumab in combinationwith 1 mg/kg ipilimumab.

TABLE 8 Overall survival in MSI-H patients. Nivolumab Nivo 3 mg/kg + 3mg/kg Ipilimumab 1 mg/kg (n = 70) (n = 30) OS rate, % (95% CI) 6 mo 75.0(58.5, 85.7) 85.1 (65.0, 94.2) mo 65.6 (48.0, 78.6) NE 12 mo 65.6 (48.0,78.6) 85.1 (65.0, 94.2) Median OS, mo (95% CI) 17.1 (8.6, NE) NE (NE,NE)

The efficacy of treatment in MSS colorectal cancer patients was measuredby ORR, PFS, and OS. Table 9 shows a summary of efficacy data in MSSpatients receiving 1 mg/kg nivolumab in combination with 3 mg/kgipilimumab compared to MSS patients receiving 3 mg/kg nivolumab incombination with 1 mg/kg ipilimumab.

TABLE 9 Summary of efficacy in MSS colorectal cancer patients. Nivolumabl mg/kg + Nivolumab 3 mg/kg + Ipilimumab 3 mg/kg Ipilimumab 1 mg/kg (n =10) (n = 10) ORR, n (%) 1 (10) 0 Median PFS, mo 2.28 (0.62, 4.40) 1.31(0.89,1.71) (95% CI) Median OS, mo 11.53 (0.62, NE) 3.73 (1.22, 5.62)(95% CI)

Treatment-related adverse events that were observed in ≥15% of MSI-Hcolorectal cancer patients included fatigue, diarrhea, pruritus, nausea,pyrexia, and vomiting. Table 10 shows the treatment-related adverseevents in ≥15% of MSI-H patients receiving 3 mg/kg nivolumab compared toMSI-H patients receiving 3 mg/kg nivolumab in combination with 1 mg/kgipilimumab.

TABLE 10 Treatment-related adverse events in ≥15% of MSI-H patients.Nivolumab Nivolumab 3 mg/kg + 3 mg/kg Ipilimumab 1 mg/kg (n = 70) (n =30) Event, n (%) Any grade Grade 3-4 Any grade Grade 3-4 Any event 41(58.6)^(a) 10 (14.3) 25 (83.3) 8 (26.7) Fatigue 13 (18.6)  1 (1.4)  6(20.0) 0 Diarrhea 10 (14.3)  1 (1.4) 13 (43.3) 0 Pruritus  8 (11.4) 0  5(16.7) 1 (3.3) Nausea  5 (7.1) 0  6 (20.0) 0 Pyrexia  3 (4.3) 0  7(23.3) 0 Vomiting  1 (1.4) 0  3 (10.0) 0 Any event leading to  4 (5.7) 2 (2.9)  4 (13.3) 4 (13.3) discontinuation ^(a)One Grade 5 event ofsudden death

Treatment-related adverse events that were observed in ≥15% of MSScolorectal cancer patients included diarrhea, asthenia, nausea, pyrexia,vomiting, fatigue, dry skin, and cough.

The results of this study demonstrated the encouraging activity ofnivolumab monotherapy in patients with MSI-H status. The combination ofnivolumab and ipilimumab also demonstrated promising preliminaryactivity. The results of this study showed that responses to nivolumabmonotherapy and to nivolumab+ipilimumab combination therapy weredurable. Nivolumab and the combination of nivolumab and ipilimumabdemonstrated tolerable safety profiles in relation to the clinicalbenefit and were consistent with observations in other solid tumors.Such results are encouraging and support the continued evaluation ofnivolumab monotherapy and nivolumab+ipilimumab combination therapy inpatients with MSI-H metastatic colorectal cancer and potentially othertumors with mismatch repair defects.

Example 2

Extended Clinical Case Study of Nivolumab+Ipilimumab in the Treatment ofPatients with Deficient DNA Mismatch Repair/High MicrosatelliteInstability Metastatic Colorectal Cancer

Further to the clinical case study described in Example 1, the efficacyand safety of nivolumab in combination with ipilimumab was investigatedin an expanded population of patients with DNA mismatchrepair-deficient/microsatellite instability-high (dMMR/MSI-H) metastaticcolorectal cancer (mCRC) who had received a first treatment dose atleast six months prior to the data cut-off. Patients were eligible forinclusion in this study based upon the following criteria: 1)histologically confirmed metastatic/recurrent colorectal cancer; 2)dMMR/MSI-H indicated by local laboratory; and 3) at least one prior lineof therapy.

FIG. 8 shows the overall study design for patients included in amonotherapy arm or combination therapy arm of treatment. Briefly,patients included in the monotherapy arm were administered 3 mg/kgnivolumab once every two weeks in Stage 1 of treatment. If there were atleast seven out of nineteen confirmed responders, enrollment wascontinued for a secondary stage of treatment. In Stage 2 of treatment,patients were administered 3 mg/kg nivolumab once every two weeks.Patients included a separate combination arm were administered 3 mg/kgnivolumab in combination with 1 mg/kg ipilimumab once every three weeksfor four doses, followed by 3 mg/kg nivolumab once every two weeks inStage 1 of treatment. If there were at least seven out of nineteenconfirmed responders, enrollment was continued for a secondary stage oftreatment. In Stage 2 of treatment, patients were administered 3 mg/kgnivolumab in combination with 1 mg/kg ipilimumab once every three weeksfor four doses, followed by 3 mg/kg nivolumab once every two weeks.Further details regarding the study design are described in Overman M etal., J Clin Oncol. 2017; 35: (suppl 4S; abstract 519) and Overman M etal., Ann Oncol. 2016; 27 (6): 149-206 (abstract 479P).

The primary endpoint of this study was to determine theinvestigator-assessed objective response rate (ORR) using RECIST 1.1.Other key endpoints included the determination of ORR by blindedindependent central review (BICR) and assessments of progression-freesurvival (PFS), overall survival (OS), and safety. Tumor imagingassessments were performed every six weeks for 24 weeks and thereafterevery 12 weeks until disease progression or discontinuation. Treatmentbeyond progression was permitted if the patient was determined by theinvestigator to be benefitting from and tolerating study therapy, andconsent was provided by the patient.

Investigator-assessed responses with nivolumab monotherapy weredetermined for 74 patients. Nivolumab monotherapy demonstrated durableresponses, sustained disease control, and a 12-month overall survival(OS) rate of 74%. The objective response rate (ORR) in patientsreceiving nivolumab monotherapy was 31%, with a median time to response(TTR) of 2.8 months. A median duration of response (DOR) was not reachedin patients receiving nivolumab monotherapy, with 83% (19/23) responsesongoing. As shown in FIG. 9, 62% of patients receiving nivolumabmonotherapy had a reduction in tumor burden from baseline. Furtherdetails regarding the nivolumab monotherapy are provided in Overman M etal., J Clin Oncol. 2017; 35: (suppl 4S; abstract 519).

The patient demographics collected for this study included median age,the number of patients under age 65, the number of male patients, thenumber of patients of a particular race, the number of patients with anECOG score of 0 or 1, the number of patients at a particular diseasestage at initial diagnosis, the number of patients with a particularclinical history of Lynch syndrome, the number of patients with aparticular BRAF or KRAS mutation status, the number of patients withtumor PD-L1 expression of ≥1% or <1% at baseline, the number of patientswith a certain number of prior lines of therapy, and the number ofpatients with prior radiotherapy. Table 11 shows the baseline patientdemographics and disease characteristics for 84 dMMR/MSI-H metastaticcolorectal cancer patients receiving combination therapy with 3 mg/kgnivolumab+1 mg/kg ipilimumab.

TABLE 11 Baseline patient demographics and disease characteristics forpatients receiving nivolumab + ipilimumab combination therapy.dMMR/MSI-H (N = 84) Age Median (range), years 57 (21-81) <65 years, n(%) 61 (73) Male, n (%) 48 (57) Race, n (%) White 77 (92) Black  2 (2)Asian  3 (4) Other  2 (2) ECOG performance status, n (%) 0 31 (37) 1 53(63) Disease stage at initial diagnosis, n (%) I-II  9 (11) III 33 (39)IV 42 (50) Clinical history of Lynch syndrome, n (%) Yes 27 (32) No 25(30) Unknown 32 (38) Mutation status, n (%) BRAF/KRAS wild type 22 (26)BRAF mutated 21 (25) KRAS mutated 30 (36) Tumor PD-L1 expressionquantifiable at baseline, n (%) ≥1% 16 (24) <1% 50 (76) Prior lines oftherapy, n (%) 0  1 (1) 1 17 (20) 2 31 (37) 3 23 (27) >4 12 (14) Priorradiotherapy, n (%) 17 (20)

Data regarding the dispositions of dMMR/MSI-H metastatic colorectalcancer patients collected in this study included the number of dosesreceived, the number of patients continuing treatment, the number ofpatients that discontinued treatment, and the number of patientsdiscontinuing treatment for a particular reason. The median time fromfirst dose to data cut-off was 8.6 months, with a range of 6.3-19.4months. Table 12 shows the patient dispositions for 84 dMMR/MSI-Hmetastatic colorectal cancer patients receiving combination therapy with3 mg/kg nivolumab+1 mg/kg ipilimumab.

TABLE 12 Patient disposition for dMMR/MSI-H metastatic colorectal cancerpatients receiving nivolumab + ipilimumab combination therapy. MMR/MSI-H(N = 84) Number of doses received, median (SD) 14.4 (10.8) Continuingtreatment, n (%)   51 (61) Discontinued treatment, n (%)   33 (39)Reasons for discontinuing treatment, n (%) Disease progression   15 (18)Treatment-related adverse event   11 (13) Death    1 (1) Adverse eventunrelated to study drug    1 (1) Lost to follow up    1 (1) Patientdecision    1 (1) Not reported/other    3 (4) SD, standard deviation

The investigator-assessed objective response rate (ORR), best overallresponse, number of patients with disease control for ≥12 weeks, mediantime to response (TTR), and median duration of response (DOR) weredetermined for dMMR/MSI-H metastatic colorectal cancer patientsreceiving nivolumab+ipilimumab combination therapy. The best overallresponse was determined by measuring the number of patients withcomplete remission, partial remission, stable disease, or progressivedisease. As shown in Table 13, an investigator-assessed ORR was achievedin 55% of patients and the disease control rate (DCR) was 79%.

TABLE 13 Summary of response and disease control for dMMR/MSI-Hmetastatic colorectal cancer patients receiving nivolumab + ipilimumabcombination therapy. dMMR/MSI-H (N = 84) ORR, n (%)  46 (55) [95%, CI][43.5, 65.7] Best overall response, n (%) CR   2 (2) PR  44 (52) SD  26(31) PD   9 (11) Not determined/reported   3 (3) Disease control for ≥12weeks,^(a) n (%)  66 (79) [95% CI] [68.3, 86.8] Median TTR, months(range) 2.8 (1.1-14.0) Median DOR, months, NR [95% CI] [NR, NR] DOR,duration of response; NR, not reached; TTR, time to response;^(a)Patients with CR, PR, or SD for ≥12 weeks.

The best reduction in target lesion size and change in tumor burden overtime was determined for dMMR/MSI-H metastatic colorectal cancer patientsreceiving nivolumab+ipilimumab combination therapy. As shown in FIG. 10,80% of patients had a reduction in tumor burden from baseline. Thepatient-specific characteristics of the change in tumor burden over timeare shown in FIG. 11. At the time of data base cut-off, 85% (39/46)responses were ongoing.

Progression-free survival (PFS) and overall survival (OS) were assessedfor dMMR/MSI-H metastatic colorectal cancer patients receivingnivolumab+ipilimumab combination therapy. The median time from the firstdose to death or the last known alive date was 8.7 months for thesepatients, with a range of 0.1 months to 20.1 months. As shown in FIG.12A, the 9-month PFS rate was 77%, while the 6-month and 12-month PFSrates were also each 77% (66.5%, 85.1% at 95% CI). The median PFS inmonths had not been reached at the time of data cut-off (11.47, Notestimable at 95% CI). As shown in FIG. 12B, the 9-month OS rate was 88%,while the 6-month OS rate was 89% (80.2%, 94.2% at 95% CI) and the12-month OS rate was 88% (78.1%, 93.1% at 95% CI). The median OS inmonths had not been reached at the time of data cut-off (Not estimable,not estimable at 95% CI).

Treatment-related adverse events (TRAEs) that were observed indMMR/MSI-H metastatic colorectal cancer patients receivingnivolumab+ipilimumab combination therapy included diarrhea, fatigue,aspartate aminotransferase increase, pyrexia, pruritis, alanineaminotransferase increase, nausea, hyperthyroidism, and hypothyroidism.All adverse events were manageable, with Grade 3/4 TRAEs reported in 29%of patients. No treatment-related deaths were reported.

The results of this study demonstrated that combination therapy withnivolumab+ipilimumab provided durable responses, sustained diseasecontrol, and encouraging survival data in pretreated patients withdMMR/MSI-H metastatic colorectal cancer. Combination therapy withnivolumab+ipilimumab also demonstrated a manageable safety profile. Suchresults are encouraging and support the continued evaluation ofcombination therapy with nivolumab+ipilimumab in patients withdMMR/MSI-H metastatic colorectal cancer and potentially other tumors.

1. A method of treating a subject afflicted with a tumor derived from acolorectal cancer, comprising administering to the subject: (i) ananti-PD-1 antibody, and (ii) an anti-CTLA-4 antibody; wherein the tumorexhibits a high degree of microsatellite instability (“MSI-H”).
 2. Themethod of claim 1, wherein the tumor is a colon cancer or a rectalcancer.
 3. The method of claim 1, wherein the tumor exhibits one or morecharacteristics selected from the group consisting of: (a) the tumorcomprises a germline alteration in at least two DNA mismatch repairgenes (“MMR genes”); (b) the tumor comprises a germline alteration in atleast 30% of five or more MMR genes; (c) at least one protein encoded byDNA MMR genes is not detected in the tumor; and (d) any combinationthereof.
 4. The method of claim 3, wherein the DNA MMR genes compriseMSH2, MLH1, MSH6, PMS2, PMS1, or any combination thereof.
 5. The methodof claim 1, wherein: (i) the subject exhibits a progression-freesurvival of at least about one month after the administration, or (ii)the subject exhibits an overall survival of at least about one monthafter the administration.
 6. (canceled)
 7. The method of claim 1,wherein the anti-PD-1 antibody is nivolumab or pembrolizumab.
 8. Themethod of claim 1, wherein the anti-CTLA-4 antibody is administered at adose ranging from at least about 0.1 mg/kg to at least about 10.0 mg/kgbody weight once about every 1, 2, 3, or 4 weeks.
 9. (canceled)
 10. Themethod of claim 1, wherein the anti-CTLA-4 antibody is ipilimumab ortremelimumab.
 11. The method of claim 1, wherein the anti-PD-1 antibodyis administered at a dose ranging from at least about 0.1 mg/kg to atleast about 10.0 mg/kg body weight once about every 1, 2, 3 or 4 weeks.12. The method of claim 1, wherein: (i) the anti-PD-1 antibody isadministered at a dose of about 1 mg/kg body weight and the anti-CTLA-4antibody is administered at a dose of about 1 mg/kg body weight; (ii)the anti-PD-1 antibody is administered at a dose of about 1 mg/kg bodyweight and the anti-CTLA-4 antibody is administered at a dose of about 3mg/kg body weight; (iii) the anti-PD-1 antibody is administered at adose of about 3 mg/kg body weight and the anti-CTLA-4 antibody isadministered at a dose of about 1 mg/kg body weight; or (iv) theanti-PD-1 antibody is administered at a dose of about 3 mg/kg bodyweight and the anti-CTLA-4 antibody is administered at a dose of about 3mg/kg body weight.
 13. The method of claim 12, wherein theadministration of the anti-PD-1 antibody and the anti-CTLA-4 antibody isfollowed by an anti-PD-1 antibody monotherapy.
 14. (canceled)
 15. A kitfor treating a patient afflicted with a tumor derived from colorectalcancer, wherein the tumor exhibits a high degree of microsatelliteinstability (“MSI-H”), the kit comprising: (a) a dosage ranging from 0.1mg/kg to 10 mg/kg body weight of an anti-PD-1 antibody; (b) a dosageranging from 0.1 mg/kg to 10 mg/kg body weight of an anti-CTLA-4antibody; and (c) instructions for using the anti-PD-1 antibody and theanti-CTLA-4 antibody in the method of claim
 1. 16. The method of claim1, wherein the anti-PD-1 antibody is administered at a dose of about 3mg/kg body weight and the anti-CTLA-4 antibody is administered at a doseof about 1 mg/kg body weight once about every 3 weeks for a total of 12weeks.
 17. The method of claim 16, wherein the administration of theanti-PD-1 antibody and the anti-CTLA-4 antibody is followed by ananti-PD-1 antibody monotherapy.
 18. The method of claim 17, wherein theanti-PD-1 antibody monotherapy is administered once about every 2 weeks.19. The method of claim 17, wherein the anti-PD-1 antibody monotherapyis administered at a dose of about 3 mg/kg once about every 2 weeks. 20.The method of claim 17, wherein the anti-PD-1 antibody monotherapy isadministered as a flat dose.
 21. The method of claim 17, wherein theanti-PD-1 antibody monotherapy is administered at a flat dose of about240 mg once about every 2 weeks.
 22. The method of claim 21, wherein theanti-PD-1 antibody is nivolumab.
 23. The method of claim 22, wherein theanti-CTLA-4 antibody is ipilimumab.